Inhibitors of MYH7B and uses thereof

ABSTRACT

The present invention provides nucleic acid inhibitors of MYH7B and compositions thereof. The present invention also provides methods of treating or preventing a cardiac disorder such as cardiac hypertrophy, myocardial infarction, or heart failure in a subject by administering to the subject an inhibitor of MYH7B. The present invention further provides methods of modulating the activity or expression of β-MHC in cardiac cells of a subject by administering to the subject an inhibitor of MYH7B.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority to U.S. Provisional Application No. 62/033,018, filed on Aug. 4, 2014, the contents of which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to inhibitors of a myosin heavy chain gene, MYH7B, and compositions thereof. The invention also provides methods for treating or preventing cardiac disorders such as cardiac hypertrophy, myocardial infarction, and heart failure by administering inhibitors of MYH7B. In particular, the invention discloses nucleic acid inhibitors of MYH7B and compositions thereof, and methods for treating or preventing cardiac disorders by inhibiting the expression or activity of MYH7B in a subject in need thereof.

BACKGROUND

Heart disease is the leading cause of a death in the United States and presents a major health risk for millions of people across the world. The cost to diagnose, treat and support patients suffering from various types of heart disease, including hypertrophic cardiomyopathy, myocardial infarction, and heart failure, is very high and puts a serious burden on the healthcare system.

Hypertrophic cardiomyopathy (HCM) is of particular concern because it is a significant cause of sudden unexpected cardiac death and is frequently asymptomatic prior to onset of cardiac arrest. HCM is characterized by a thickening of myocardial cells, which can affect ventricular function and give rise to cardiac arrhythmias. HCM is a primary genetic disease of the heart, resulting from a dominant mutation in a number of sarcomeric genes, of which 8 have been extensively studied (Teekakirikul et al., “Hypertrophic cardiomyopathy: translating cellular cross talk into therapeutics,” J. Cell. Biol., 2012, 199(3), 417-421). Of these dominant mutations in HCM, roughly 40% are missense mutations in the β-myosin gene, MYH7. Genetic and pharmacologic studies have shown reductions in expression of this dominant allele, even to a small degree, can have robust phenotypic effects (Jiang et al., “Allele-specific silencing of mutant MYH6 transcripts in mice suppresses hypertrophic cardiomyopathy,” Science, 2013, 342(6154), 111-114).

Myosin is the major contractile protein of cardiac and skeletal muscle cells. Cardiac muscle contraction depends on the expression and relative ratios of two myosin heavy chain (MHC) proteins, α-MHC (MYH6) and β-MHC (MYH7). In rodents, α-MHC, a fast-twitching MHC, is the predominant myosin isoform in the adult heart, whereas β-MHC, a slow-twitching MHC, is predominantly expressed in the developing heart and is downregulated after birth (Morkin, E., “Control of cardiac myosin heavy chain gene expression,” Microsc. Res. Tech., 2000, 50, 522-531). In contrast, in human heart, the β-MHC isoform is heavily expressed and the α-MHC isoform accounts for less than 8% of total ventricular MHC (Miyata et al., “Myosin heavy chain isoform expression in the failing and nonfailing human heart,” Circ. Res., 2000, 86(4):386-90). Irrespective of the differences in the expression of alpha- and beta-myosins in various species, studies indicate that expression of beta-myosin is upregulated in cardiac disorders in these species including humans, rats, and rabbits. For example, in failing adult mouse hearts, a shift from the normally predominant alpha-MHC toward beta-MHC is often observed (Harada et al., Circulation, 1999, 100, 2093-2099). Similarly, in rats, congestive heart failure was associated with increased expression of beta-myosin and decreased expression of alpha-MHC. Consistent with rodent studies, beta-myosin expression was upregulated and alpha-myosin expression was significantly down-regulated in failing human hearts (Miyata et al., “Myosin heavy chain isoform expression in the failing and nonfailing human heart”, Circ Res. 2000 Mar. 3; 86(4):386-90). These studies show that although the expression of alpha- and beta-myosins is species-dependent, downregulating the expression of beta-myosin is likely to play a cardioprotective role in various species. For example, blunting the increase in β-MHC expression and increasing α-MHC expression has shown to be cardioprotective in rabbits (James et al., “Forced expression of alpha-myosin heavy chain in the rabbit ventricle results in cardioprotection under cardiomyopathic conditions,” Circulation, 2005, 111(18), 2339-2346).

The genes encoding α-MHC (MYH6), β-MHC (MYH7), and a related myosin, MYH7B, also encode a family of intronic miRNAs, miR-208a, miR-208b, and miR-499, respectively. These three miRs share sequence homology and are called the “MyomiRs” (van Rooji et al., “A family of microRNAs encoded by myosin genes governs myosin expression and muscle performance,” Dev. Cell, 2009, 17, 662-673). MyomiRs have been shown to control pathological cardiac remodeling, muscle myosin content, myofiber identity, and muscle performance (Liu and Olson, “MicroRNA regulatory networks in cardiovascular development,” Dev. Cell, 2010, 510-525).

Although the role of α-MHC, β-MHC, and myomiRs (miR-208a, miR-208b, and miR-499) in cardiac and muscle development have been extensively studied, the role of the third myosin, MYH7B, is largely unknown. The MYH7B gene is expressed in skeletal muscle, heart, and in a subset of cells in the brain where it regulates synapse structure and function in the brain (Yeung et al., “Myh7b/miR-499 gene expression is transcriptionally regulated by MRFs and Eos,” Nucleic Acids Res., 2012, 40(15):7303-18). A recent report suggests that MYH7b protein is detected in a minor fibre population in extraocular muscles, corresponding to slow-tonic fibres, and in bag fibres of muscle spindles (Rossi et al., “Two novel/ancient myosins in mammalian skeletal muscles: MYH14/7b and MYH15 are expressed in extraocular muscles and muscle spindles”, J Physiol., Jan. 15, 2010; 588(Pt 2):353-64). However, in human heart, MYH7B gene is considered to undergo non-productive splicing, and result in a RNA that may not encode a functional MYH7B protein (Bell et al., “Uncoupling of expression of an intronic microRNA and its myosin host gene by exon skipping,” Mol. Cell. Biol., 30, 1937-1945). Interestingly, the non-productive splicing of MYH7B mRNA in heart cells was not associated with altered expression of intronic miR-499. Thus, until the present invention, MYH7B was considered to be a non-functioning carrier of miR-499 in cardiac cells (Gerald Dorn, II, “MicroRNAs: redefining mechanisms in cardiac disease,” J. Cardiovasc. Pharmacol., 2010, 56(6), 589-595). Moreover, unlike miR-499, MYH7B is considered not to play an important role in cardiac and skeletal muscle biology.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the surprising discovery that MYH7B regulates the expression of β-MHC in cardiac cells and thus regulates cardiac contractility and stress-induced cardiac remodeling. Specifically, the inhibition of MYH7B results in the down-regulation of β-MHC mRNA and protein in cardiac cells. Accordingly, in one embodiment, the present invention provides inhibitors of MYH7B, compositions thereof, and methods for modulating the expression and/or activity of β-MHC in a subject in need thereof comprising administering to the subject the inhibitor of MYH7B. In one embodiment, administration of an inhibitor of MYH7B to the subject downregulates the expression or activity of MYH7B in cardiac cells of the subject following administration. In an embodiment, administration of an inhibitor of MYH7B to the subject downregulates the expression or activity of β-MHC (MYH7) in cardiac cells of the subject following administration. In another embodiment, administration of an inhibitor of MYH7B to the subject upregulates the expression or activity of α-MHC (MYH6) in cardiac cells of the subject following administration. In yet another embodiment, administration of an inhibitor of MYH7B to the subject induces a “switch” in the myosins whereby the expression or activity of β-MHC is downregulated and the expression or activity of α-MHC is upregulated in the subject following administration. In one embodiment, administration of an inhibitor of MYH7B to the subject does not change the expression or activity of miR-208a, miR-208b, or miR-499 in cardiac cells of the subject.

In another embodiment, the present invention provides compositions and methods for treating or preventing cardiac disorders such as pathologic cardiac hypertrophy, myocardial infarction, heart failure, or hypertrophic cardiomyopathy in a subject in need thereof comprising administering an inhibitor of MYH7B to the subject. In one embodiment, the subject having a cardiac disorder has increased levels of β-MHC in cardiac cells compared to a healthy subject. In another embodiment, the subject having a cardiac disorder has an altered ratio of β-MHC/α-MHC in cardiac cells compared to a healthy subject. In one embodiment, the administration of an inhibitor of MYH7B to a subject having a cardiac disorder inhibits the expression or activity of MYH7B and/or the expression or activity of MYH7 in cardiac cells of the subject following administration. In another embodiment, the administration of a MYH7B inhibitor to a subject having a cardiac disorder restores the β-MHC/α-MHC ratio in cardiac cells of the subject fully or partially to normal levels following administration. In one embodiment, the cardiac disorder is cardiac hypertrophy, myocardial infarction, heart failure, or hypertrophic cardiomyopathy.

In one embodiment, an inhibitor of MYH7B is a nucleic acid inhibitor selected from an antisense oligonucleotide, an aptamer, a ribozyme, a small interfering RNA (siRNA), or a small hairpin RNA (shRNA). In another embodiment, an inhibitor of MYH7B is an antibody or a binding fragment thereof that specifically binds to MYH7B protein. In a particular embodiment, an inhibitor of MYH7B is an antisense oligonucleotide that comprises a sequence that is at least partially complementary to a MYH7B coding sequence. The antisense oligonucleotides used in the compositions and methods of the invention may have a length of from about 6 to about 22 nucleotides. In some embodiments, the antisense oligonucleotides comprise one or more chemical modifications, such as sugar, backbone, and/or base modifications.

In one embodiment, an antisense oligonucleotide inhibitor of MYH7B has a length of 8 to 18 nucleotides, and wherein the sequence of the antisense oligonucleotide is substantially complementary to the sequence of SEQ ID NO: 6.

In some embodiments, an antisense oligonucleotide inhibitor of MYH7B has a length of 8-18 nucleotides and the sequence of the antisense oligonucleotide is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4300-4335.

In some other embodiments, the antisense oligonucleotide inhibitor of MYH7B has a length of 12-18 nucleotides and the sequence of the antisense oligonucleotide is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4300-4317.

In still other embodiments, the antisense oligonucleotide inhibitor of MYH7B has a length of 12-18 nucleotides and the sequence of the antisense oligonucleotide is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4316-4333.

In one embodiment, the antisense oligonucleotide inhibitor of MYH7B has a length of 14 nucleotides and the sequence of the antisense oligonucleotide is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4302-4315.

In another embodiment, the antisense oligonucleotide inhibitor of MYH7B has a length of 14 nucleotides and the sequence of the antisense oligonucleotide is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4318-4331.

In some embodiments, antisense oligonucleotide inhibitors of the invention contain at least one modified nucleotide. The modified nucleotide may include a sugar, base, and/or a backbone modification.

In one embodiment, the modified nucleotide is a locked nucleotide. In some embodiments, the antisense oligonucleotide inhibitor contains one to six locked nucleotides. In some embodiments, at least the first three nucleotides at the 5′ end of the antisense oligonucleotide inhibitor are locked nucleotides. In another embodiment, at least the first three nucleotides at the 3′ end of the antisense oligonucleotide inhibitor are locked nucleotides.

In some embodiments, at least the first three nucleotides at the 5′ end of the antisense oligonucleotide inhibitor are locked or non-locked ribonucleotides. In some embodiments, the at least first three nucleotides at the 3′ end of the antisense oligonucleotide inhibitor are locked or non-locked ribonucleotides.

In one embodiment, the antisense oligonucleotide inhibitor contains at least one deoxyribonucleotide. In other embodiments, the antisense oligonucleotide inhibitor contains two to eight deoxyribonucleotides.

In various embodiments, the antisense oligonucleotide inhibitor of MYH7B may include a sugar modification selected from the group consisting of 2′-O, 4′-C methylene bridge, 2′-O, 4′-C ethylene bridge, 2′-CH₂—NH—CH₂-4′ bridge, 2′-deoxy, 2′-O-alkyl, and 2′-halo modifications.

In various embodiments, the antisense oligonucleotide inhibitor of MYH7B may include a backbone modification. In one embodiment, the antisense oligonucleotide inhibitor contains at least one phosphorothioate linkage. In another embodiment, the antisense oligonucleotide inhibitor contains two or more phosphorothioate linkages. In still other embodiments, the antisense oligonucleotide is fully phosphorothioate linked.

In one embodiment, the antisense oligonucleotide inhibitor of MYH7B further comprises a peptide or a sugar moiety connected through a linker. The linker could be a 3′thiol-modified-C₃-disulfide linker, (C₆)₂ disulfide linker, or 1-3 phosphodiester linkages.

In certain embodiments, the antisense oligonucleotide inhibitor of MYH7B comprises a sequence selected from Tables 1-5.

In one embodiment, the antisense oligonucleotide inhibitor of MYH7B comprises the sequence of 5′-lTslTslGsdAsdTsdCsdTsdTsdGsdGsdCslCslTslC-3′ (SEQ ID NO: 146) or 5′-lCslTslGsdCsdAsdGsdCsdTsdCsdCsdTslCslCslA-3′ (SEQ ID NO: 148).

The invention also provides pharmaceutical compositions comprising an inhibitor of MYH7B and a pharmaceutically acceptable excipient. In one embodiment, the inhibitor of MYH7B is an antisense oligonucleotide. In one aspect, the pharmaceutical composition further comprises a second therapeutic agent, wherein the second therapeutic agent is an antisense oligonucleotide inhibitor of miR-208a, miR-208b, miR-499, miR-15a, miR-15b, miR-16, miR-195, or a mixture thereof.

The invention further provides a method for treating or preventing pathologic cardiac hypertrophy, myocardial infarction, or heart failure in a subject in need thereof, comprising administering to the subject an antisense oligonucleotide inhibitor of MYH7B. In one embodiment, the pathologic cardiac hypertrophy is hypertrophic cardiomyopathy. In some embodiments, the methods according to the invention may comprise administering a second cardiac therapeutic agent such as an antisense oligonucleotide inhibitor of miR-208a, miR-208b, miR-499, miR-15a, miR-15b, miR-16, miR-195, or combinations thereof.

In some embodiments, the invention provides a method of treating a subject who is at risk for pathologic cardiac hypertrophy. In one embodiment, the subject at risk for pathologic cardiac hypertrophy has a mutation in a beta myosin heavy chain gene.

In one aspect, administration of the antisense oligonucleotide inhibitor of MYH7B reduces the expression or activity of MYH7B in cardiac cells of the subject following administration. In another aspect, administration of the antisense oligonucleotide inhibitor of MYH7B reduces the expression of beta myosin heavy chain in cardiac cells of the subject following administration. In yet another aspect, administration of the antisense oligonucleotide inhibitor of MYH7B does not significantly alter the expression of miR-499, miR-208a, and/or miR-208b in cardiac cells of the subject following administration.

In some embodiments, the invention provides a method for treating or preventing pathologic cardiac hypertrophy, myocardial infarction, or heart failure in a subject in need thereof, comprising administering to the subject an inhibitor of MYH7B. In various embodiments, the inhibitor of MYH7B is a nucleic acid inhibitor selected from an antisense oligonucleotide, an aptamer, a ribozyme, a small interfering RNA, or a small hairpin RNA.

In one embodiment, the nucleic acid inhibitor of MYH7B is a small interfering RNA or a small hairpin RNA comprising a double-stranded region of about 10 to about 30 nucleotides, said double-stranded region comprising (i) a first RNA strand having a sequence that is at least 70% identical to a sequence of the human Myh7b gene and (ii) a second RNA strand that is partially, substantially, or fully complementary to the first RNA strand. In one aspect, the first RNA strand of the siRNA or shRNA has a sequence that is at least 70% identical to a sequence of 5′-GAGGCCAAGATCAA-3′ (SEQ ID NO: 4). In another aspect, the first RNA strand of the siRNA or shRNA has a sequence that is at least 70% identical to a sequence of 5′-TGGAGGAGCTGCAG-3′ (SEQ ID NO: 5).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows levels of MYH7B mRNA in human iPS cardiomyocytes 48 hours after transfection with antisense oligonucleotides to MYH7B. FIG. 1B shows levels of MYH7 (13-MHC) mRNA in human iPS cardiomyocytes 48 hours after transfection with antisense oligonucleotides to MYH7B.

FIG. 2A shows levels of miR-208a in human iPS cardiomyocytes 48 hours after transfection with antisense oligonucleotides to MYH7B. FIG. 2B shows levels of miR-499 in human iPS cardiomyocytes 48 hours after transfection with antisense oligonucleotides to MYH7B. FIG. 2C shows levels of miR-208b in human iPS cardiomyocytes 48 hours after transfection with antisense oligonucleotides to MYH7B.

FIG. 3A shows levels of MYH7 (β-MHC) mRNA in control and HCM-R663H cardiomyocytes 48 hours after transfection with antisense oligonucleotides to MYH7B. FIG. 3B shows levels of MYH7 (β-MHC) mRNA in control and HCM-R663H cardiomyocytes 168 hours (1 week) after transfection with antisense oligonucleotides to MYH7B.

FIG. 4 shows a Western blot analysis of MYH7 (β-MHC) or GAPDH from control and HCM iPS cardiomyocytes at 96 hours post-treatment.

FIG. 5 shows levels of MYH7B mRNA in human iPS cardiomyocytes 48 hrs after transfection with test ASO compounds to MYH7B.

FIG. 6A shows levels of MYH7B mRNA in human iPS cardiomyocytes 168 hrs after passive administration of 1, 5, or 10 μM test ASO compounds (SEQ ID NOs. 92, 146, 32, 46, and 30) targeted to MYH7B. FIG. 6B shows levels of MYH7 (β-MHC) mRNA in human iPS cardiomyocytes 168 hrs after passive administration of 1, 5, or 10 μM the test ASO compounds. FIG. 6C shows levels of MYH6 (α-MHC) mRNA in human iPS cardiomyocytes 168 hrs after passive administration of 1, 5, or 10 μM of the test ASO compounds.

FIG. 7 shows levels of MYH7B, MYH7 (β-MHC), and MYH6 (α-MHC) mRNAs in human iPS cardiomyocytes 168 hrs after passive administration of 5 μM test ASO having the sequence of SEQ ID NO: 148.

FIG. 8 shows levels of MYH7B mRNA in left ventricles of rats injected with 25 mg/kg of test ASOs per day for three consecutive days and sacrificed 48 hours after the last dose.

FIG. 9A shows levels of MYH7B mRNA in left ventricles of rabbits treated with ASO compounds comprising the sequence of SEQ ID NOs: 146 and 148. FIG. 9B shows levels of MYH7B mRNA in right ventricles of rabbits treated with ASO compounds comprising the sequence of SEQ ID NOs: 146 and 148. FIG. 9C shows levels of MYH7 (β-MHC) mRNA in left ventricles of rabbits treated with ASO compounds comprising the sequence of SEQ ID NOs: 146 and 148. FIG. 9D shows levels of MYH7 (β-MHC) mRNA in right ventricles of rabbits treated with ASO compounds comprising the sequence of SEQ ID NOs: 146 and 148.

FIG. 10A shows levels of MYH7B mRNA in human iPS cardiomyocytes 48 hrs after passive administration of ENA-modified ASOs based on the compound comprising the sequence of SEQ ID NO: 146. FIG. 10B shows levels of MYH7B mRNA in human iPS cardiomyocytes 48 hrs after passive administration of 5-methyl cytidine and 2′-O-methyl modified ASOs of the compound comprising the sequence of SEQ ID NO: 146. FIG. 10C shows levels of MYH7B mRNA in human iPS cardiomyocytes 48 hrs after passive administration of backbone-modified ASOs of the compound comprising the sequence of SEQ ID NO: 146.

FIGS. 11A-11D show levels of MYH7B mRNA in human iPS cardiomyocytes 48 hrs after passive administration of test ASO compounds directed to MYH7B.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have found that MYH7B, which was considered until now to act merely as a non-functional carrier of miR-499, plays an important role in regulating the expression of β-MHC in cardiac cells. Particularly, the inventors have found that the inhibition of MYH7B down-regulates the expression of β-MHC mRNA and protein in cardiac cells. Thus, the present invention provides MYH7B as a new target for therapeutic intervention for cardiac disorders, particularly cardiac muscle disorders.

The present invention provides agents that inhibit the expression or activity of MYH7B. In various embodiments, the agent that inhibits MYH7B is a nucleic acid inhibitor targeted to MYH7B gene, mRNA, or pre-mRNA or an antibody or a binding fragment thereof targeted to MYH7B protein. In certain embodiments, a nucleic acid inhibitor of MYH7B is selected from an antisense oligonucleotide, an aptamer, a ribozyme, a small interfering RNA, or a small hairpin RNA.

In one embodiment, an inhibitor of MYH7B is an antisense oligonucleotide (“ASO”). In the context of the present invention, the term “antisense oligonucleotide” or “ASO” is used broadly and encompasses an oligomer comprising ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides or a combination thereof that inhibits the information transfer from gene to protein. The term “antisense oligonucleotide” or “ASO” as used herein also includes oligomers comprising ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides or a combination thereof that facilitate transcriptional arrest; disruption of mRNA synthesis from pre-mRNA at various stages including capping, splicing, transport from nucleus to cytoplasm; degradation of mRNA such as that mediated by gapmer ASOs or other RNAse H-inducing ASOs; translational arrest where the ASO binds to target mRNA and makes it unavailable for translation; or steric blocking of target mRNA by hybridizing to it. The term “antisense oligonucleotide” as used herein also includes antisense oligonucleotide conjugates that comprise oligomers containing natural and/or modified nucleotides conjugated to or connected to a peptide or a sugar through a linker. The peptide or the sugar moiety and the linkers that can be used to prepare antisense oligonucleotide conjugates are described below in more detail.

In various embodiments, antisense oligonucleotides useful for inhibiting the activity of MYH7B are about 5 to about 25 nucleotides in length, about 6 to about 22 nucleotides in length, about 10 to about 30 nucleotides in length, about 12 to about 20 nucleotides in length, or about 20 to about 25 nucleotides in length. In certain embodiments, antisense oligonucleotides targeting MYH7B are about 8 to about 18 nucleotides in length, in other embodiments about 12 to about 18 nucleotides in length, and in still other embodiments about 12 to about 16 nucleotides in length. In some embodiments, antisense oligonucleotides targeting MYH7B are about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.

In some embodiments, antisense oligonucleotide inhibitors directed to MYH7B are about 50 to about 150 nucleotides in length, about 60 to about 140 nucleotides in length, about 70 to about 130 nucleotides in length, about 80 to about 120 nucleotides in length, about 90 to about 110 nucleotides in length, or about 100 to about 120 nucleotides in length. In one embodiment, antisense oligonucleotide inhibitors are about 100 to about 120 nucleotides in length. In certain embodiments, antisense oligonucleotide inhibitors targeting MYH7B are about 80, 90, 100, 110, or 120 nucleotides in length.

Antisense oligonucleotides targeting MYH7B comprise a sequence that is at least partially or substantially complementary to a MYH7B sequence (DNA, mRNA, or pre-mRNA) to hybridize to MYH7B under physiological conditions and inhibit the expression or activity of MYH7B in the cells of a subject. For instance, in some embodiments, antisense oligonucleotides comprise a sequence that is at least partially complementary to a MYH7B gene, mRNA or pre-mRNA sequence, e.g. at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to a MYH7B gene, mRNA or pre-mRNA sequence. In some embodiments, the antisense oligonucleotide can be substantially complementary to a MYH7B gene, mRNA or pre-mRNA sequence, that is at least about 90%, 95%, 96%, 97%, 98%, or 99% complementary to a target polynucleotide sequence. In one embodiment, the antisense oligonucleotide comprises a sequence that is 100% or completely complementary to a MYH7B gene, mRNA or pre-mRNA sequence. In certain embodiments, the antisense oligonucleotide targeting MYH7B comprises a sequence that is at least partially, substantially, or completely complementary to the sequence of the human MYH7B gene. In some embodiments, the antisense oligonucleotide targeting MYH7B comprises a sequence that is at least partially, substantially, or completely complementary to the coding sequence of the human MYH7B gene (Genebank Accession No. NM_020884.4; SEQ ID NO: 6).

Human MYH7B coding sequence (SEQ ID NO: 6):    1 taggcttaaa gagcaggcgg ggcagagacc catacctttg gcgctgagtc gaggccagga   61 aggggcggtc gatgagaggg cggccttagc attaggagtg cgtcggctgc accctggcgg  121 atggaccttg ggaaggaggg gaggggacca tcggggtcgg cggggagcta cgggttcccc  181 tcttccaaga cgggtgccaa gtgatgagta tcaggtgtct atggtgagat ttggggagga  241 tcccagacct taacagccct ttggttgaaa cctctcacaa tgtggcagag atggggtttt  301 gatgtgttgc ccaggctggt cttgaactcc tgagcttaag tgatgtgtcc gcctgggcct  361 cccaaagtgc tggaattaca gacatgagtg gcaataaaag gggtagcaga gcttcctgcc  421 ctcaccgtgg tgccgagtgc ctgctgcctt gggccgcctt gaacctccag ggtttccagc  481 tcctcctcct tcaccccagt gccactgcca tgatggatgt gagtgaactt ggggagtctg  541 cccgctacct ccgccagggc taccaggaga tgacgaaggt gcacactatc ccatgggacg  601 ggaagaagcg agtctgggtg cctgatgaac aggacgccta cgtggaggcc gaggtcaagt  661 cggaggctac cgggggcaga gtcaccgtgg agaccaaaga ccagaaggtg ctgatggtgc  721 gtgaagccga gctgcagccc atgaacccgc ctcgcttcga cttactggag gacatggcca  781 tgatgacgca cctgaacgag gcctctgtgc tgcacaacct gcgccagcgc tatgcccgct  841 ggatgatcta tacctactca ggcctcttct gtgtcaccat caacccctac aaatggctcc  901 cagtctatac ggcctccgta gtggctgctt acaagggaaa gcgccgctca gattccccgc  961 cccatatata tgcggtggcg gacaacgcct acaacgacat gctgcgcaac cgagacaacc 1021 agtccatgct gatcaccgga gagtcggggg ccggtaagac ggttaacacc aagcgggtca 1081 ttcagtactt tgccatcgtc gctgccctgg gagacgggcc gggcaagaag gcccaatttc 1141 tggcaacaaa gacggggggc acccttgagg atcaaatcat cgaggccaac cctgccatgg 1201 aggcctttgg caacgccaag accctgagga atgataactc ctcccgcttt ggcaagttca 1261 tccgcattca ctttggtccc tctgggaagc tggcatccgc ggatattgac agctatctcc 1321 tggagaagtc gcgggtgatc ttccagttgc ctggtgagcg cagctaccat gtctactacc 1381 agatcctctc agggaggaag ccagagctgc aggacatgct gcttctgtct atgaacccct 1441 atgactacca cttctgcagc cagggcgtca tcaccgtgga caacatgaat gatggggagg 1501 agctcatcgc caccgaccat gccatggaca tcctaggctt cagcgtggat gagaaatgtg 1561 cctgctataa gatcgtgggc gccctcctgc actttggcaa catgaagttc aagcagaagc 1621 agcgggagga gcaggcggag gccgatggca ctgagagtgc tgacaaggct gcctacctga 1681 tgggggtcag cagtggggac ctcctcaaag gccttttgca cccccgggtg cgtgtaggga 1741 acgagtacgt gaccaagggc cagagtgtgg agcaggtggt gtttgctgtg ggggctctgg 1801 ccaaggccac ctatgaccgg ctgttcaggt ggctggtgtc tcggatcaac cagaccctgg 1861 acacaaagct gccccggcag ttcttcatcg gggttctgga catcgctggg tttgagatct 1921 ttgagttcaa cagcttcgaa cagctgtgca tcaacttcac caatgagaaa ttgcagcagt 1981 tcttcaacca gcacatgttt gtgctggagc aggaggagta caagcgggag ggcatcgact 2041 gggtcttcat cgacttcggc cttgacctgc agccttgcat cgacctcatc gagaagccac 2101 tgggcatcct gtccatcctg gaggaggaat gcatgttccc caaggcctca gacgccagct 2161 tccgggccaa gctctacgac aaccacgcgg ggaagtcacc caatttccag cagcctcggc 2221 ctgacaagaa gcgcaagtac caggcccact tcgaggtggt ccactacgca ggcgtggtgc 2281 cttacagcat tgtgggctgg ctggagaaaa acaaggatcc cctgaatgag accgtggtcc 2341 ccatcttcca gaagtcacag aataggctcc tggcgactct ctatgagaat tatgcgggct 2401 cctgctccac tgagcccccc aagtctgggg tgaaagagaa gcgtaagaag gcagcatcgt 2461 tccagacggt gtcccagctg cacaaggaga acctcaacaa gctgatgacc aacctgcggg 2521 ccacacagcc ccacttcgtc cgctgcattg tccccaacga gaacaaaacc ccaggggtca 2581 tggatgcctt cttggtgcta caccagctgc gctgcaatgg ggtcctggag gggatccgga 2641 tctgccgcca agggttcccc aacaggttgc tctacaccga cttccggcag cggtaccgta 2701 tcctgaaccc cagtgccatc ccggatgaca ccttcatgga cagcaggaag gccacagaga 2761 aactgctggg ctcgctggac ttggatcaca cccagtacca gtttggccac accaaggtgt 2821 tcttcaaggc tgggcttcta ggcgtcctgg aagagctccg tgaccagcgc ctggccaagg 2881 tgctgacgct gctgcaggcg cggagccgtg gccgcctcat gcgccttgag taccagcgcc 2941 tgctgggagg cagggatgcg ctgttcacca tccagtggaa catccgtgcc ttcaatgccg 3001 tcaagaactg gtcatggatg aagctctttt tcaagatgaa gccgctgctg cgctcggcgc 3061 aggctgagga ggagctggcg gccctgcggg cagagctgcg ggggttgcga ggggcgctgg 3121 ctgcggccga ggccaagcgc caggaactgg aggagacgca cgtcagcatc acccaggaga 3181 agaatgacct ggccctgcag ctgcaggctg agcaggacaa cctggcagat gccgaggagc 3241 gctgccactt gctgatcaag tccaaggtgc agctggaggg gaaggtgaag gagctgagtg 3301 agcggctgga ggatgaggag gaggtgaacg ctgacctggc cgcccgccgg cgcaagctgg 3361 aggacgagtg cacggagctc aagaaggaca ttgatgacct ggagctgaca ctggccaaag 3421 ctgagaagga gaagcaagcc actgagaaca aggtgaagaa cctgacggaa gagatggctg 3481 cgctggacga gtcagtggcc cggctgacca aggagaagaa ggcgttgcag gaggcccacc 3541 aacaggccct gggtgacctg caggccgagg aggaccgtgt gagcgcgctg accaaggcca 3601 agctccggct ggagcaacag gtggaggacc tggaatgctc cctggagcag gagaagaagc 3661 tgcgcatgga cacggagcgg gccaagcgca agctggaggg tgacctgaag ctgacgcagg 3721 agtcggtggc tgatgctgct caagacaagc agcagctgga ggagaagctc aagaagaagg 3781 actccgagct gagccagctg agcctgcggg tggaagacga gcagctcttg ggggcccaga 3841 tgcagaagaa gatcaaggag ctgcaggctc gggcggagga gctggaagag gagctggagg 3901 cagagcgggc agcccgggcc cgcgtggaga agcagcgtgc agaggcggcg cgggagctgg 3961 aggagctgag cgagcggctg gaggaggcag gcggcgcatc cgcggggcag cgcgagggct 4021 gccgcaagcg ggaggcggag ctggggaggc tgcggcggga gctggaggag gcggcgctgc 4081 ggcacgaggc cacagtggcg gcactgcggc gcaagcaggc ggagggcgcg gcggagctgg 4141 gggagcaggt ggacagcctg cagcgggtgc ggcagaagct ggagaaggag aagagtgagc 4201 tgcgcatgga ggtggacgac ctggctgcca acgtggagac tctgacccgc gccaaggcca 4261 gtgcagagaa gctgtgccgg acctatgagg atcagctaag cgaggccaag atcaaggtgg 4321 aggagctgca gcggcagctg gcggacgcaa gcacgcagcg tgggcgacta cagacggaaa 4381 gcggggagct gagtcgcctg ctagaggaga aggagtgtct gatcagtcag ctgagccgtg 4441 gaaaggccct ggccgcccaa agcctggaag agttgcggcg ccagctagag gaggaaagca 4501 aggccaagag tgccctggcc cacgccgtgc aggctctgcg gcacgactgt gacctcctgc 4561 gggagcaaca cgaggaggag gctgaggccc aggctgagct gcagcggctg ctgtccaagg 4621 ccaatgccga ggtggcccag tggaggagca agtacgaagc agatgccatc cagaggaccg 4681 aggagctgga ggaggccaaa aaaaagctgg cactgcggct gcaggaggca gaggagggcg 4741 tggaggctgc caacgccaag tgctcatcgt tggagaaggc caagctgcgg ctacagacag 4801 agtcagagga tgtaaccctg gagctggagc gggcgacctc agcagctgct gcgctggaca 4861 agaagcagcg gcacttggaa cgggcactgg aggaacggcg gcggcaggag gaggagatgc 4921 agcgggagct ggaggcggca cagagggagt cccgtggcct gggcaccgag ctcttccggc 4981 tgcggcacgg ccacgaggag gcacttgaag ccctggagac gctcaagegg gagaacaaga 5041 acctgcagga ggagatcagc gacctcacag accaggtgag tctcagtggg aagagcatcc 5101 aggaactgga gaaaaccaag aaggcgctgg aaggcgagaa gagtgagatc caggctgcac 5161 tggaggaggc agagggggcc ctggagctgg aggagaccaa gacgctgcgg atccagctgg 5221 agctctccca ggtcaaagca gaagtggacc ggaagctggc agagaaagac gaggagtgcg 5281 ctaacctgag gcgcaaccac cagcgagctg tggagtccct gcaggcctcc ctggatgcag 5341 agacacgggc ccgcaatgag gcgctgcggc tcaagaagaa gatggagggt gacctcaacg 5401 acctggagct gcagctgggc catgccaccc gtcaggccac agaggcccag gctgccacgc 5461 ggctgatgca ggcacagctc aaggaggagc aggcagggcg ggacgaggag cagcggctgg 5521 cagctgagct ccacgagcag gcgcaggctc tggagcgccg ggcctcgctg ctggctgcgg 5581 agctggagga gctgcgggct gccctggagc agggcgagcg cagccggcga ctggcagagc 5641 aggagctttt ggaggccacc gagcgcctca accttctgca ttcgcagaac acaggcctcc 5701 taaaccagaa gaagaagctg gaggcggact tggcccagct gagcggggag gtggaggagg 5761 ctgcacagga gaggcgggag gctgaggaga aggccaaaaa ggccatcact gatgcggcca 5821 tgatggccga ggagctgaag aaggagcagg acacaagtgc acacctggaa cggatgaaga 5881 agacgctgga gcagacggtg cgcgagctcc aggcccgcct tgaggaggca gaacaggccg 5941 ccctccgtgg cgggaagaag caggtgcaga agctggaggc caaggtacgg gagctggagg 6001 ctgagcttga tgcagagcag aagaagcacg ccgaggccct taagggcgtg cgcaagcatg 6061 agcgccgtgt caaggagctc gcataccagg ccgaggagga caggaagaac ctggctcgca 6121 tgcaggacct ggtggacaag ctgcagagca aggtcaagag ctacaagcgc cagtttgagg 6181 aggcggagca gcaggccaac accaacctgg ccaagtatcg caaggcccag cacgagctgg 6241 atgatgcgga ggagcgggca gacatggcgg aaacccaggc caacaagctg cgggcacgga 6301 cccgggacgc cctgggcccc aagcacaagg agtgacggcc tgaccccctg ggctctaaag 6361 aggaatgtct gctgttgcac atctggctga ggccacctgc cccgatcctg ccatctctgc 6421 atcgccccct gctgccttca gccttccctg ggccctgaat aaacaccaca gccagtttcc 6481 ttctcattct tttctttggg gttcaggagg aaaaacacag tcctagggac aaaagccagg 6541 tccacagcag tcatttttaa aataaagtta tttaatagtc tccaaaaaaa aaaaaaaaaa 6601 aa

It is understood that the sequence of the antisense oligonucleotide inhibitor is considered to be complementary to a MYH7B gene, mRNA or pre-mRNA sequence even if the antisense oligonucleotide sequence includes a modified nucleotide instead of a naturally-occurring nucleotide. For example, if a MYH7B gene, mRNA or pre-mRNA sequence comprises a guanosine nucleotide at a specific position, the antisense oligonucleotide inhibitor may comprise a modified cytidine nucleotide, such as a locked cytidine nucleotide or 2′-fluoro-cytidine, at the corresponding position.

In one embodiment, the invention provides an antisense oligonucleotide inhibitor of MYH7B, wherein said antisense oligonucleotide has a length of 8 to 18 nucleotides, and wherein the sequence of the antisense oligonucleotide is substantially complementary to the sequence of SEQ ID NO: 6.

In one embodiment, the antisense oligonucleotide inhibitor has a length of 8 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4300-4335.

In certain embodiments, the antisense oligonucleotide inhibitor has a length of 12-18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4300-4317, 4300-4316, 4301-4316, 4301-4315, 4302-4317, 4302-4316, or 4302-4315. In one embodiment, the antisense oligonucleotide inhibitor has a length of 12-14 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4302-4315. In some embodiments, the antisense oligonucleotide inhibitor has a length of 14 nucleotides and the sequence of the antisense oligonucleotide is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4302-4315. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lTslTslGsdAsdTsdCsdTsdTsdGsdGsdCslCslTslC-3′ (SEQ ID NO: 146), where l=locked nucleic acid modification, d=deoxynucleotide, and s=phosphorothioate linkage.

In certain embodiments, the antisense oligonucleotide inhibitor has a length of 12-18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4316-4333, 4316-4332, 4317-4332, 4317-4331, 4318-4333, 4318-4332, or 4318-4331. In one embodiment, the antisense oligonucleotide inhibitor has a length of 12-14 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4318-4331. In some embodiments, the antisense oligonucleotide has a length of 14 nucleotides and the sequence of the antisense oligonucleotide is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4318-4331. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lCslTslGsdCsdAsdGsdCsdTsdCsdCsdTslCslCslA-3′ (SEQ ID NO: 148).

In some embodiments, the antisense oligonucleotide inhibitor has a length of 8 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 1762-1783. In one embodiment, the antisense oligonucleotide inhibitor has a length of 12-18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 1762-1779, 1763-1779, 1764-1781, 1765-1780, or 1766-1779. In one embodiment, the antisense oligonucleotide inhibitor has a length of 12-14 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 1764-1777 or 1766-1779. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lAslCslCsdTsdGsdCsdTsdCsdCsdAsdCslAslCslT-3′ (SEQ ID NO: 62). In another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lCslCslAsdCsdCsdTsdGsdCsdTsdCsdCslAslCslA-3′ (SEQ ID NO: 64).

In some embodiments, the antisense oligonucleotide inhibitor has a length of 8 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 511-538. In one embodiment, the antisense oligonucleotide inhibitor has a length of 12-18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 511-532, 512-530, 513-526, 514-528, 514-527, 515-530, 515-529, or 515-528. In one embodiment, the antisense oligonucleotide inhibitor has a length of 12-14 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 513-526, 514-527, or 515-528. In yet another embodiment, the antisense oligonucleotide inhibitor has a length of 12-18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 521-538, 522-538, 522-537, 523-538, 523-537, or 523-536. In yet another embodiment, the antisense oligonucleotide inhibitor has a length of 12-14 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 522-537 or 523-536. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lTslCslAsmdCsdTsmdCsdAsmdCsdAsdTsmdCslCslAslT-3′, (SEQ ID NO: 184), where and =5-methylcytosine. In another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lTslTslCsdAsmdCsdTsmdCsdAsmdCsdAsdTslCslCslA-3′ (SEQ ID NO: 186). In yet another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lGslTslTsmdCsdAsmdCsdTsmdCsdAsmdCsdAslTslCslC-3′ (SEQ ID NO: 188). In still other embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lCslTslCsmdCsmdCsmdCsdAsdAsdGsdTsdTslCslAslC-3′ (SEQ ID NO: 204).

In certain embodiments, the antisense oligonucleotide inhibitor has a length of 8 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 1640-1701. In one embodiment, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 1643-1660, 1648-1665, 1653-1670, or 1694-1711. In some embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 14 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 1645-1658, 1650-1663, 1655-1668, or 1696-1709. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lAslCslTsmdCsdTsmdCsdAsdGsdTsdGsmdCslCslAslT-3′ (SEQ ID NO: 230). In another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lTslCslAsdGsmdCsdAsmdCsdTsmdCsdTsmdCslAslGslT-3′ (SEQ ID NO: 238). In yet another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lCslCslTsdTsdGsdTsmdCsdAsdGsmdCsdAslCslTslC-3′ (SEQ ID NO: 244). In yet another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lTslTslTsdGsdAsdGsdGsdAsdGsdGsdTslCslCslC-3′ (SEQ ID NO: 272).

In some embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4280-4300. In one embodiment, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4280-4297, 4282-4295, 4283-4300 or 4283-4296. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lCslTslGsdAsdTsmdCsmdCsdTsmdCsdAsdTslAslGslG-3′ (SEQ ID NO: 304). In another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lGslCslTsdGsdAsdTsmdCsmdCsdTsmdCsdAslTslAslG-3′ (SEQ ID NO: 306).

In some embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 688-705, 689-706, 690-707, or 690-703. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lTslGslGsdTsmdCsdTsdTsdTsdGsdGsdTslCslTslC-3′ (SEQ ID NO: 344). In other embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 890-907, 892-909, or 892-905. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lGslAslCsdTsdGsdGsdGsdAsdGsmdCsmdCslAslTslT-3′ (SEQ ID NO: 350).

In some embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 1217-1234, 1218-1235, 1219-1236, or 1219-1232. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lAslTslTsmdCsmdCsdTsmdCsdAsdGsdGsdGslTslCslT-3′ (SEQ ID NO: 362). In other embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 1224-1241, 1225-1242, 1226-1243, or 1226-1239. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lAslGslTsdTsdAsdTsmdCsdAsdTsdTsmdCslCslTslC-3′ (SEQ ID NO: 370).

In some embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 1415-1441. In some embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 1415-1432, 1416-1433, 1417-1434, or 1417-1430. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lAslGslAsmdCsdAsdGsdAsdAsdGsmdCsdAslGslCslA-3′ (SEQ ID NO: 396). In some embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 1422-1439, 1423-1440, 1424-1441, or 1424-1437. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lGslGslTsdTsmdCsdAsdTsdAsdGsdAsmdCslAslGslA-3′ (SEQ ID NO: 410).

In some embodiments, the antisense oligonucleotide inhibitor has a length of 8 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 2348-2371. In one embodiment, the antisense oligonucleotide inhibitor has a length of 12-18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 2348-2365, 2349-2364, 2348-2361, 2349-2366, 2350-2365, 2351-2366, 2352-2367, 2352-2365, 2353-2370, 2354-2369, or 2354-2368. In one embodiment, the antisense oligonucleotide inhibitor has a length of 12-14 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 2349-2362 or 2350-2363. In yet another embodiment, the antisense oligonucleotide inhibitor has a length of 12-14 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 2350-2363 or 2351-2364. In yet another embodiment, the antisense oligonucleotide inhibitor has a length of 12-14 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 2352-2365. In yet another embodiment, the antisense oligonucleotide inhibitor has a length of 12-14 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 2353-2366 or 2354-2367. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lAslTslTsmdCsdTsdGsdTsdGsdAsmdCsdTslTslCslT-3′ (SEQ ID NO: 474). In another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lTslAslTsdTsmdCsdTsdGsdTsdGsdAsmdCslTslTslC-3′ (SEQ ID NO: 476). In yet another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lCslTslAsdTsdTsmdCsdTsdGsdTsdGsdAslCslTslT-3′ (SEQ ID NO: 478). In still other embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lGslCslCsdTsdAsdTsdTsmdCsdTsdGsdTslGslAslC-3′ (SEQ ID NO: 482).

In some embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 2442-2462. In certain embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 14 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 2444-2457 or 2445-2458. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lAslTslGsmdCsdTsdGsmdCsmdCsdTsdTsmdCslTslTslA-3′ (SEQ ID NO: 508). In another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lGslAslTsdGsmdCsdTsdGsmdCsmdCsdTsdTslCslTslT-3′ (SEQ ID NO: 510).

In some other embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4408-4432. In various embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4408-4425, 4409-4426, 4410-4425, 4411-4428, 4411-4427, 4412-4429, 4412-4428, or 4413-4430. In one embodiment, the antisense oligonucleotide inhibitor has a length of 12 to 16 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4409-4424 or 4410-4425. In another embodiment, the antisense oligonucleotide inhibitor has a length of 12 to 16 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4411-4426 or 4412-4427. In yet another embodiment, the antisense oligonucleotide inhibitor has a length of 12 to 16 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4413-4428. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lAslTslCsdAsdGsdAsmdCsdAsmdCsdTsmdCslCslTslT-3′ (SEQ ID NO: 570). In another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lTslGslAsdTsmdCsdAsdGsdAsmdCsdAsmdCslTslCslC-3′ (SEQ ID NO: 572). In yet another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lCslTslGsdAsdTsmdCsdAsdGsdAsmdCsdAslCslTslC-3′ (SEQ ID NO: 574).

In some embodiments, the antisense oligonucleotide targeting MYH7B comprises a sequence that is at least partially complementary or completely complementary to the sequence of human MYH7B gene from nucleotides 1182 to 1213 in SEQ ID NO: 6. In other embodiments, the antisense oligonucleotide targeting MYH7B comprises a sequence that is at least partially complementary or completely complementary to the sequence of human MYH7B gene from nucleotides 1365 to 1393 in SEQ ID NO: 6.

In one embodiment, the antisense oligonucleotide targeting MYH7B has a sequence of 5′-GTGAATGCGGATGAA-3′ (SEQ ID NO: 1). In another embodiment, the antisense oligonucleotide of MYH7B has a sequence of 5′-GAAGTGGTAGTCATA-3′ (SEQ ID NO: 2). In yet another embodiment, the antisense oligonucleotide of MYH7B has a sequence of 5′-lGslTslGsdAsdAsdTsdGsdCsdGsdGsdAsdTsdGslAslA-3′ (SEQ ID NO: 3).

The term “about” as used herein encompasses variations of +/−10% and more preferably +/−5%, as such variations are appropriate for practicing the present invention.

In one embodiment, the antisense oligonucleotide inhibitor of MYH7B contains at least one backbone modification, such as at least one phosphorothioate, morpholino, or phosphonocarboxylate internucleotide linkage (see, for example, U.S. Pat. Nos. 6,693,187 and 7,067,641, which are herein incorporated by reference in their entireties). In some embodiments, the antisense oligonucleotide inhibitor of MYH7B contains two or more phosphorothioate linkages. In certain embodiments, the antisense oligonucleotide inhibitor of MYH7B is fully phosphorothioate-linked. Alternatively, the antisense oligonucleotides can comprise peptide nucleic acids (PNAs), which contain a peptide-based backbone rather than a sugar-phosphate backbone.

In one embodiment, the antisense oligonucleotide inhibitor of MYH7B contains at least one modified nucleotide such as a locked nucleotide or a nucleotide containing other sugar, base, and/or backbone modifications. The terms “locked nucleotide,” “locked nucleic acid unit,” “locked nucleic acid residue,” “LNA unit”, “bridged nucleic acid,” “bridged nucleotide,” “BNA” may be used interchangeably throughout the disclosure and refer to a bicyclic nucleoside/nucleotide analogue. Specifically, a “bridged nucleic acid” or “bridged nucleotide” refers to a nucleotide containing a bridge at the 2′,4′-position or at the 3′,4′-position of the sugar residue. For instance, suitable antisense oligonucleotide inhibitors can be comprised of one or more “conformationally constrained” or 2′,4′-bridged nucleotide modifications that confer enhanced thermal stability to complexes formed between the oligonucleotide containing bridged nucleotides and their complementary target strand. In one embodiment, antisense oligonucleotide inhibitors contain a 2′-O, 4′-C-methylene bridge ribonucleoside (structure A), conventionally known as “locked nucleotide” or “LNA”. In another embodiment, antisense oligonucleotide inhibitors contain at least one 2′,4′-C-bridged 2′ deoxyribonucleoside (CDNA, structure B). See, e.g., U.S. Pat. No. 6,403,566 and Wang et al. (1999) Bioorganic and Medicinal Chemistry Letters, Vol. 9: 1147-1150, both of which are herein incorporated by reference in their entireties. In yet another embodiment, antisense oligonucleotide inhibitors contain at least one modified nucleoside having the structure shown in structure C. In yet another embodiment, antisense oligonucleotide inhibitors contain at least one 2′O, 4′-C-ethylene bridge ribonucleoside (structure D). A nucleic acid or an oligonucleotide or a nucleotide containing the 2′O, 4′-C-ethylene bridge is also known as ethylene-bridged nucleic acid (ENA). In still another embodiment, antisense oligonucleotide inhibitors contain at least one 2′-CH₂—NH—CH₂-4′-bridged nucleotide, also known as “amino-2′-C-Bridged Bicyclic Nucleotide” or “amino-CBBN”. The antisense oligonucleotide inhibitors targeting MYH7B can contain combinations of bridged nucleotides described above or other modified nucleotides, and unmodified ribonucleotides or deoxyribonucleotides.

When referring to substituting a DNA or RNA nucleotide by its corresponding bridged or locked nucleotide in the context of the present invention, the terms “corresponding bridged nucleotide” or “corresponding locked nucleotide” are intended to mean that the DNA/RNA nucleotide has been replaced by a bridged or locked nucleotide containing the same naturally-occurring nitrogenous base as the DNA/RNA nucleotide that it has replaced or the same nitrogenous base that is chemically modified. For example, the corresponding bridged or locked nucleotide of a DNA nucleotide containing the nitrogenous base C may contain the same nitrogenous base C or the same nitrogenous base C that is chemically modified, such as 5-methylcytosine.

The terms “non-bridged nucleotide” or “non-locked nucleotide” refer to a nucleotide different from a bridged or locked-nucleotide, i.e. the terms “non-bridged nucleotide” or “non-locked nucleotide” include a DNA nucleotide, an RNA nucleotide as well as a modified nucleotide where a base and/or sugar is modified except that the modification is not a bridge or locked modification.

In one embodiment, the antisense oligonucleotide inhibitor of MYH7B contains at least one locked nucleotide. In some embodiments, the antisense oligonucleotide inhibitor of MYH7B contains one to six locked nucleotides. In one embodiment, at least the first three nucleotides from the 3′ end of the antisense oligonucleotide inhibitor are locked nucleotides. In another embodiment, at least the first three nucleotides from the 5′ end of the antisense oligonucleotide inhibitor are locked nucleotides.

In certain embodiments, the antisense oligonucleotide inhibitor contains one to six natural or modified ribonucleotides. In one embodiment, at least the first three nucleotides from the 3′ end of the antisense oligonucleotide inhibitor are natural or modified ribonucleotides. In another embodiment, at least the first three nucleotides from the 5′ end of the antisense oligonucleotide inhibitor are natural or modified ribonucleotides. In some embodiments, the first three nucleotides from the 3′ end of the antisense oligonucleotide inhibitor are locked ribonucleotides. In other embodiments, the first three nucleotides from the 5′ end of the antisense oligonucleotide inhibitor are locked ribonucleotides.

Oligonucleotide inhibitors of the present invention may include modified nucleotides that have a base modification or substitution. The natural or unmodified bases are the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C), thymine (T) and uracil (U). Modified bases, also referred to as heterocyclic base moieties, include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (including 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines), 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. In one embodiment, antisense oligonucleotide inhibitors of the present invention comprise one to five 5-methylcytidine or 5-methyldeoxycytidine nucleotides. In certain embodiments, antisense oligonucleotide inhibitors comprise one or more bridged nucleic acid modifications (e.g. LNA, ENA, etc.) in combination with a base modification (e.g. 5-methyl cytidine).

Oligonucleotide inhibitors of the present invention may include nucleotides with modified sugar moieties. Representative modified sugars include carbocyclic or acyclic sugars, sugars having substituent groups at one or more of their 2′, 3′ or 4′ positions and sugars having substituents in place of one or more hydrogen atoms of the sugar. In certain embodiments, the sugar is modified by having a substituent group at the 2′ position. In additional embodiments, the sugar is modified by having a substituent group at the 3′ position. In other embodiments, the sugar is modified by having a substituent group at the 4′ position. It is also contemplated that a sugar may have a modification at more than one of those positions, or that an oligonucleotide inhibitor may have one or more nucleotides with a sugar modification at one position and also one or more nucleotides with a sugar modification at a different position.

Sugar modifications contemplated in the oligonucleotide inhibitors of the present invention include, but are not limited to, a substituent group selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted with C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. In one embodiment, the modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, which is also known as 2′-O-(2-methoxyethyl) or 2′-MOE), that is, an alkoxyalkoxy group. Another modification includes 2′-dimethylaminooxyethoxy, that is, a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), that is, 2′-O—CH₂—O—CH₂—N(CH₃)₂.

Additional sugar substituent groups include allyl (—CH₂—CH═CH₂), —O-allyl, methoxy (—O—CH₃), aminopropoxy (—OCH₂CH₂CH₂NH₂), and fluoro (F). Sugar substituent groups on the 2′ position (2′-) may be in the arabino (up) position or ribo (down) position. One 2′-arabino modification is 2′-F. Other similar modifications may also be made at other positions on the sugar moiety, particularly the 3′ position of the sugar on the 3′ terminal nucleoside or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. In certain embodiments, the sugar modification is a 2′-O-alkyl (e.g. 2′-O-methyl, 2′-O-methoxyethyl), 2′-halo (e.g., 2′-fluoro, 2′-chloro, 2′-bromo), and 4′ thio modifications.

Other modifications of oligonucleotide inhibitors to enhance stability and improve efficacy, such as those described in U.S. Pat. No. 6,838,283, which is herein incorporated by reference in its entirety, are known in the art and are suitable for use in the methods of the invention. For instance, to facilitate in vivo delivery and stability, the oligonucleotide inhibitor can be linked to a steroid, such as cholesterol moiety, a vitamin, a fatty acid, a carbohydrate or glycoside, a peptide, or other small molecule ligand at its 3′ end. In some embodiments, the antisense oligonucleotide inhibitor is attached to a peptide to enhance the uptake of the inhibitor by target cells. For instance, in some embodiments, the 3′ end of an antisense oligonucleotide inhibitor is attached to a peptide having the sequence “WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 788)”. This peptide is described in McGuire et al. (“In vitro selection of a peptide with high selectivity for cardiomyocytes in vivo”, J. Mol. Bio, 2004, vol. 342, 171-182), which is hereby incorporated by reference herein. In one embodiment, the peptide is attached to the antisense oligo via a linker such as “thiolC3,” whereby a thioether bond is formed between the SH group of the linker and the oligo and the maleimide on the peptide. In some embodiments, the 5′end of an antisense oligonucleotide inhibitor could be attached to a sugar moiety directly or via a linker. The linker could be a C6-disulfide linker or a 1-3 phosphodiester linker such as a phosphodiester dTdT linker.

In various embodiments, the antisense oligonucleotide of MYH7B has a sequence selected from Tables 1-5.

TABLE 1 Starting  nucleotide of target sequence in SEQ ID NO: 6 Sequence(5′-3′) Sequence(5′-3′) with modifications  733 GTTCATGGGCTGCA lGslTslTsdCsdAsdTsdGsdGsdGsdCsdTslGslCslA (SEQ ID NO: 7) (SEQ ID NO: 8)  734 GGTTCATGGGCTGC lGslGslTsdTsdCsdAsdTsdGsdGsdGsdCslTslGslC (SEQ ID NO: 9) (SEQ ID NO: 10)  735 GGGTTCATGGGCTG lGslGslGsdTsdTsdCsdAsdTsdGsdGsdGslCslTslG (SEQ ID NO: 11) (SEQ ID NO: 12)  867 ATGGTGACACAGAA lAslTslGsdGsdTsdGsdAsdCsdAsdCsdAslGslAslA (SEQ ID NO: 13) (SEQ ID NO: 14) 1020 ATCAGCATGGACTG lAslTslCsdAsdGsdCsdAsdTsdGsdGsdAslCslTslG (SEQ ID NO: 15) (SEQ ID NO: 16) 1061 GCTTGGTGTTAACC lGslCslTsdTsdGsdGsdTsdGsdTsdTsdAslAslCslC (SEQ ID NO: 17) (SEQ ID NO: 18) 1188 TCCATGGCAGGGTT lTslCslCsdAsdTsdGsdGsdCsdAsdGsdGslGslTslT (SEQ ID NO: 19) (SEQ ID NO: 20) 1189 CTCCATGGCAGGGT lCslTslCsdCsdAsdTsdGsdGsdCsdAsdGslGslGslT (SEQ ID NO: 21) (SEQ ID NO: 22) 1190 CCTCCATGGCAGGG lCslCslTsdCsdCsdAsdTsdGsdGsdCsdAslGslGslG (SEQ ID NO: 23) (SEQ ID NO: 24) 1191 GCCTCCATGGCAGG lGslCslCsdTsdCsdCsdAsdTsdGsdGsdCslAslGslG (SEQ ID NO: 25) (SEQ ID NO: 26) 1305 AGATAGCTGTCAAT lAslGslAsdTsdAsdGsdCsdTsdGsdTsdCslAslAslT (SEQ ID NO: 27) (SEQ ID NO: 28) 1306 GAGATAGCTGTCAA lGslAslGsdAsdTsdAsdGsdCsdTsdGsdTslCslAslA (SEQ ID NO: 29) (SEQ ID NO: 30) 1307 GGAGATAGCTGTCA lGslGslAsdGsdAsdTsdAsdGsdCsdTsdGslTslCslA (SEQ ID NO: 31) (SEQ ID NO: 32) 1308 AGGAGATAGCTGTC lAslGslGsdAsdGsdAsdTsdAsdGsdCsdTslGslTslC (SEQ ID NO: 33) (SEQ ID NO: 34) 1309 CAGGAGATAGCTGT lCslAslGsdGsdAsdGsdAsdTsdAsdGsdCslTslGslT (SEQ ID NO: 35) (SEQ ID NO: 36) 1310 CCAGGAGATAGCTG lCslCslAsdGsdGsdAsdGsdAsdTsdAsdGslCslTslG (SEQ ID NO: 37) (SEQ ID NO: 38) 1311 TCCAGGAGATAGCT lTslCslCsdAsdGsdGsdAsdGsdAsdTsdAslGslCslT (SEQ ID NO: 39) (SEQ ID NO: 40) 1443 CAGAAGTGGTAGTC lCslAslGsdAsdAsdGsdTsdGsdGsdTsdAslGslTslC (SEQ ID NO: 41) (SEQ ID NO: 42) 1445 TGCAGAAGTGGTAG lTslGslCsdAsdGsdAsdAsdGsdTsdGsdGslTslAslG (SEQ ID NO: 43) (SEQ ID NO: 44) 1446 CTGCAGAAGTGGTA lCslTslGsdCsdAsdGsdAsdAsdGsdTsdGslGslTslA (SEQ ID NO: 45) (SEQ ID NO: 46) 1647 GCACTCTCAGTGCC lGslCslAsdCsdTsdCsdTsdCsdAsdGsdTslGslCslC (SEQ ID NO: 47) (SEQ ID NO: 48) 1662 TAGGCAGCCTTGTC lTslAslGsdGsdCsdAsdGsdCsdCsdTsdTslGslTslC (SEQ ID NO: 49) (SEQ ID NO: 50) 1755 ACACTCTGGCCCTT lAslCslAsdCsdTsdCsdTsdGsdGsdCsdCslCslTslT (SEQ ID NO: 51) (SEQ ID NO: 52) 1758 TCCACACTCTGGCC lTslCslCsdAsdCsdAsdCsdTsdCsdTsdGslGslCslC (SEQ ID NO: 53) (SEQ ID NO: 54) 1759 CTCCACACTCTGGC lCslTslCsdCsdAsdCsdAsdCsdTsdCsdTslGslGslC (SEQ ID NO: 55) (SEQ ID NO: 56) 1760 GCTCCACACTCTGG lGslCslTsdCsdCsdAsdCsdAsdCsdTsdCslTslGslG (SEQ ID NO: 57) (SEQ ID NO: 58) 1761 TGCTCCACACTCTG lTslGslCsdTsdCsdCsdAsdCsdAsdCsdTslCslTslG (SEQ ID NO: 59) (SEQ ID NO: 60) 1764 ACCTGCTCCACACT lAslCslCsdTsdGsdCsdTsdCsdCsdAsdCslAslCslT (SEQ ID NO: 61) (SEQ ID NO: 62) 1766 CCACCTGCTCCACA lCslCslAsdCsdCsdTsdGsdCsdTsdCsdCslAslCslA (SEQ ID NO: 63) (SEQ ID NO: 64) 1767 ACCACCTGCTCCAC lAslCslCsdAsdCsdCsdTsdGsdCsdTsdCslCslAslC (SEQ ID NO: 65) (SEQ ID NO: 66) 1912 CTCAAAGATCTCAA lCslTslCsdAsdAsdAsdGsdAsdTsdCsdTslCslAslA (SEQ ID NO: 67) (SEQ ID NO: 68) 1913 ACTCAAAGATCTCA lAslCslTsdCsdAsdAsdAsdGsdAsdTsdCslTslCslA (SEQ ID NO: 69) (SEQ ID NO: 70) 1914 AACTCAAAGATCTC lAslAslCsdTsdCsdAsdAsdAsdGsdAsdTslCslTslC (SEQ ID NO: 71) (SEQ ID NO: 72) 2037 ATGAAGACCCAGTC lAslTslGsdAsdAsdGsdAsdCsdCsdCsdAslGslTslC (SEQ ID NO: 73) (SEQ ID NO: 74) 2038 GATGAAGACCCAGT lGslAslTsdGsdAsdAsdGsdAsdCsdCsdCslAslGslT (SEQ ID NO: 75) (SEQ ID NO: 76) 2253 TAGTGGACCACCTC lTslAslGsdTsdGsdGsdAsdCsdCsdAsdCslCslTslC (SEQ ID NO: 77) (SEQ ID NO: 78) 2481 AGGTTCTCCTTGTG lAslGslGsdTsdTsdCsdTsdCsdCsdTsdTslGslTslG (SEQ ID NO: 79) (SEQ ID NO: 80) 2482 GAGGTTCTCCTTGT lGslAslGsdGsdTsdTsdCsdTsdCsdCsdTslTslGslT (SEQ ID NO: 81) (SEQ ID NO: 82) 2483 TGAGGTTCTCCTTG lTslGslAsdGsdGsdTsdTsdCsdTsdCsdCslTslTslG (SEQ ID NO: 83) (SEQ ID NO: 84) 2484 TTGAGGTTCTCCTT lTslTslGsdAsdGsdGsdTsdTsdCsdTsdCslCslTslT (SEQ ID NO: 85) (SEQ ID NO: 86) 2485 GTTGAGGTTCTCCT lGslTslTsdGsdAsdGsdGsdTsdTsdCsdTslCslCslT (SEQ ID NO: 87) (SEQ ID NO: 88) 2488 CTTGTTGAGGTTCT lCslTslTsdGsdTsdTsdGsdAsdGsdGsdTslTslCslT (SEQ ID NO: 89) (SEQ ID NO: 90) 2518 GGGCTGTGTGGCCC lGslGslGsdCsdTsdGsdTsdGsdTsdGsdGslCslCslC (SEQ ID NO: 91) (SEQ ID NO: 92) 2738 CCTTCCTGCTGTCC lCslCslTsdTsdCsdCsdTsdGsdCsdTsdGslTslCslC (SEQ ID NO: 93) (SEQ ID NO: 94) 2740 GGCCTTCCTGCTGT lGslGslCsdCsdTsdTsdCsdCsdTsdGsdCslTslGslT (SEQ ID NO: 95) (SEQ ID NO: 96) 2961 TGGATGGTGAACAG lTslGslGsdAsdTsdGsdGsdTsdGsdAsdAslCslAslG (SEQ ID NO: 97) (SEQ ID NO: 98) 2962 CTGGATGGTGAACA lCslTslGsdGsdAsdTsdGsdGsdTsdGsdAslAslCslA (SEQ ID NO: 99) (SEQ ID NO: 100) 2964 CACTGGATGGTGAA lCslAslCsdTsdGsdGsdAsdTsdGsdGsdTslGslAslA (SEQ ID NO: 101) (SEQ ID NO: 102) 2966 TCCACTGGATGGTG lTslCslCsdAsdCsdTsdGsdGsdAsdTsdGslGslTslG (SEQ ID NO: 103) (SEQ ID NO: 104) 2967 TTCCACTGGATGGT lTslTslCsdCsdAsdCsdTsdGsdGsdAsdTslGslGslT (SEQ ID NO: 105) (SEQ ID NO: 106) 3186 TGCAGGGCCAGGTC lTslGslCsdAsdGsdGsdGsdCsdCsdAsdGslGslTslC (SEQ ID NO: 107) (SEQ ID NO: 108) 3187 CTGCAGGGCCAGGT lCslTslGsdCsdAsdGsdGsdGsdCsdCsdAslGslGslT (SEQ ID NO: 109) (SEQ ID NO: 110) 3255 ACCTTGGACTTGAT lAslCslCsdTsdTsdGsdGsdAsdCsdTsdTslGslAslT (SEQ ID NO: 111) (SEQ ID NO: 112) 3257 GCACCTTGGACTTG lGslCslAsdCsdCsdTsdTsdGsdGsdAsdCslTslTslG (SEQ ID NO: 113) (SEQ ID NO: 114) 3258 TGCACCTTGGACTT lTslGslCsdAsdCsdCsdTsdTsdGsdGsdAslCslTslT (SEQ ID NO: 115) (SEQ ID NO: 116) 3259 CTGCACCTTGGACT lCslTslGsdCsdAsdCsdCsdTsdTsdGsdGslAslCslT (SEQ ID NO: 117) (SEQ ID NO: 118) 3260 GCTGCACCTTGGAC lGslCslTsdGsdCsdAsdCsdCsdTsdTsdGslGslAslC (SEQ ID NO: 119) (SEQ ID NO: 120) 3261 AGCTGCACCTTGGA lAslGslCsdTsdGsdCsdAsdCsdCsdTsdTslGslGslA (SEQ ID NO: 121) (SEQ ID NO: 122) 4253 CTGCACTGGCCTTG lCslTslGsdCsdAsdCsdTsdGsdGsdCsdCslTslTslG (SEQ ID NO: 123) (SEQ ID NO: 124) 4254 TCTGCACTGGCCTT lTslCslTsdGsdCsdAsdCsdTsdGsdGsdCslCslTslT (SEQ ID NO: 125) (SEQ ID NO: 126) 4255 CTCTGCACTGGCCT lCslTslCsdTsdGsdCsdAsdCsdTsdGsdGslCslCslT (SEQ ID NO: 127) (SEQ ID NO: 128) 4256 TCTCTGCACTGGCC lTslCslTsdCsdTsdGsdCsdAsdCsdTsdGslGslCslC (SEQ ID NO: 129) (SEQ ID NO: 130) 4257 TTCTCTGCACTGGC lTslTslCsdTsdCsdTsdGsdCsdAsdCsdTslGslGslC (SEQ ID NO: 131) (SEQ ID NO: 132) 4258 CTTCTCTGCACTGG lCslTslTsdCsdTsdCsdTsdGsdCsdAsdCslTslGslG (SEQ ID NO: 133) (SEQ ID NO: 134) 4259 GCTTCTCTGCACTG lGslCslTsdTsdCsdTsdCsdTsdGsdCsdAslCslTslG (SEQ ID NO: 135) (SEQ ID NO: 136) 4260 AGCTTCTCTGCACT lAslGslCsdTsdTsdCsdTsdCsdTsdGsdCslAslCslT (SEQ ID NO: 137) (SEQ ID NO: 138) 4261 CAGCTTCTCTGCAC lCslAslGsdCsdTsdTsdCsdTsdCsdTsdGslCslAslC (SEQ ID NO: 139) (SEQ ID NO: 140) 4262 ACAGCTTCTCTGCA lAslCslAsdGsdCsdTsdTsdCsdTsdCsdTslGslCslA (SEQ ID NO: 141) (SEQ ID NO: 142) 4263 CACAGCTTCTCTGC lCslAslCsdAsdGsdCsdTsdTsdCsdTsdCslTslGslC (SEQ ID NO: 143) (SEQ ID NO: l44) 4302 TTGATCTTGGCCTC lTslTslGsdAsdTsdCsdTsdTsdGsdGsdCslCslTslC (SEQ ID NO: 145) (SEQ ID NO: l46) 4318 CTGCAGCTCCTCCA lCslTslGsdCsdAsdGsdCsdTsdCsdCsdTslCslCslA (SEQ ID NO: 147) (SEQ ID NO: 148) 4717 TGCCTCCTGCAGCC lTslGslCsdCsdTsdCsdCsdTsdGsdCsdAslGslCslC (SEQ ID NO: 149) (SEQ ID NO: 150) 4773 AGCTTGGCCTTCTC lAslGslCsdTsdTsdGsdGsdCsdCsdTsdTslCslTslC (SEQ ID NO: 151) (SEQ ID NO: 152) 4904 TCTCCTCCTCCTGC lTslCslTsdCsdCsdTsdCsdCsdTsdCsdCslTslGslC (SEQ ID NO: 153) (SEQ ID NO: 154) 6099 AGGTTCTTCCTGTC lAslGslGsdTsdTsdCsdTsdTsdCsdCsdTslGslTslC (SEQ ID NO: 155) (SEQ ID NO: 156) 6101 CCAGGTTCTTCCTG lCslCslAsdGsdGsdTsdTsdCsdTsdTsdCslCslTslG (SEQ ID NO: 157) (SEQ ID NO: 158) 6102 GCCAGGTTCTTCCT lGslCslCsdAsdGsdGsdTsdTsdCsdTsdTslCslCslT (SEQ ID NO: 159) (SEQ ID NO: 160) 6103 AGCCAGGTTCTTCC lAslGslCsdCsdAsdGsdGsdTsdTsdCsdTslTslCslC (SEQ ID NO: 161) (SEQ ID NO: 162) 6104 GAGCCAGGTTCTTC lGslAslGsdCsdCsdAsdGsdGsdTsdTsdCslTslTslC (SEQ ID NO: 163) (SEQ ID NO: 164) 6142 GACCTTGCTCTGCA lGslAslCsdCsdTsdTsdGsdCsdTsdCsdTslGslCslA (SEQ ID NO: 165) (SEQ ID NO: 166) 6143 TGACCTTGCTCTGC lTslGslAsdCsdCsdTsdTsdGsdCsdTsdCslTslGslC (SEQ ID NO: 167) (SEQ ID NO: 168) 6144 TTGACCTTGCTCTG lTslTslGsdAsdCsdCsdTsdTsdGsdCsdTslCslTslG (SEQ ID NO: 169) (SEQ ID NO: 170) 6146 TCTTGACCTTGCTC lTslCslTsdTsdGsdAsdCsdCsdTsdTsdGslCslTslC (SEQ ID NO: 171) (SEQ ID NO: 172) 6149 AGCTCTTGACCTTG lAslGslCsdTsdCsdTsdTsdGsdAsdCsdCslTslTslG (SEQ ID NO: 173) (SEQ ID NO: 174) 6151 GTAGCTCTTGACCT lGslTslAsdGsdCsdTsdCsdTsdTsdGsdAslCslCslT (SEQ ID NO: 175) (SEQ ID NO: 176) 6220 GTGCTGGGCCTTGC lGslTslGsdCsdTsdGsdGsdGsdCsdCsdTslTslGslC (SEQ ID NO: 177) (SEQ ID NO: 178) 6273 TTGTTGGCCTGGGT lTslTslGsdTsdTsdGsdGsdCsdCsdTsdGslGslGslT (SEQ ID NO: 179) (SEQ ID NO: 180) 6277 CAGCTTGTTGGCCT lCslAslGsdCsdTsdTsdGsdTsdTsdGsdGslCslCslT (SEQ ID NO: 181) (SEQ ID NO: 182) GTGAATGCGGATGAA lGslTslGsdAsdAsdTsdGsdCsdGsdGsdAsdTsdGslA (SEQ ID NO: 1) slA (SEQ ID NO: 3) l = 1ocked nuc1eotide; d = deoxynuc1eotide; s = phosphorotioate 1inkage.

TABLE 2 Starting nucleotide of target sequence in SEQ ID NO: 6 Sequence (5′-3′) Sequence (5′-3′) with modifications  513 TCACTCACATCCAT lTslCslAsmdCsdTsmdCsdAsmdCsdAsdTsmdCslCslAslT (SEQ ID NO: 183) (SEQ ID NO: 184)  514 TTCACTCACATCCA lTslTslCsdAsmdCsdTsmdCsdAsmdCsdAsdTslCslCslA (SEQ ID NO: 185) (SEQ ID NO: 186)  515 GTTCACTCACATCC lGslTslTsmdCsdAsmdCsdTsmdCsdAsmdCsdAslTslCslC (SEQ ID NO: 187) (SEQ ID NO: 188)  516 AGTTCACTCACATC lAslGslTsdTsmdCsdAsmdCsdTsmdCsdAsmdCslAslTslC (SEQ ID NO: 189) (SEQ ID NO: 190)  517 AAGTTCACTCACAT lAslAslGsdTsdTsmdCsdAsmdCsdTsmdCsdAslCslAslT (SEQ ID NO: 191) (SEQ ID NO: 192)  518 CAAGTTCACTCACA lCslAslAsdGsdTsdTsmdCsdAsmdCsdTsmdCslAslCslA (SEQ ID NO: 193) (SEQ ID NO: 194)  519 CCAAGTTCACTCAC lCslCslAsdAsdGsdTsdTsmdCsdAsmdCsdTslCslAslC (SEQ ID NO: 195) (SEQ ID NO: 196)  520 CCCAAGTTCACTCA lCslCslCsdAsdAsdGsdTsdTsmdCsdAsmdCslTslCslA (SEQ ID NO: 197) (SEQ ID NO: 198)  521 CCCCAAGTTCACTC lCslCslCsmdCsdAsdAsdGsdTsdTsmdCsdAslCslTslC (SEQ ID NO: 199) (SEQ ID NO: 200)  522 TCCCCAAGTTCACT lTslCslCsmdCsmdCsdAsdAsdGsdTsdTsmdCslAslCslT (SEQ ID NO: 201) (SEQ ID NO: 202)  523 CTCCCCAAGTTCAC lCslTslCsmdCsmdCsmdCsdAsdAsdGsdTsdTslCslAslC (SEQ ID NO: 203) (SEQ ID NO: 204)  524 ACTCCCCAAGTTCA lAslCslTsmdCsmdCsmdCsmdCsdAsdAsdGsdTslTslCslA (SEQ ID NO: 205) (SEQ ID NO: 206)  525 GACTCCCCAAGTTC lGslAslCsdTsmdCsmdCsmdCsmdCsdAsdAsdGslTslTslC (SEQ ID NO: 207) (SEQ ID NO: 208)  526 AGACTCCCCAAGTT lAslGslAsmdCsdTsmdCsmdCsmdCsmdCsdAsdAslGslTslT (SEQ ID NO: 209) (SEQ ID NO: 210)  527 CAGACTCCCCAAGT lCslAslGsdAsmdCsdTsmdCsmdCsmdCsmdCsdAslAslGslT (SEQ ID NO: 211) (SEQ ID NO: 212)  528 GCAGACTCCCCAAG lGslCslAsdGsdAsmdCsdTsmdCsmdCsmdCsmdCslAslAslG (SEQ ID NO: 213) (SEQ ID NO: 214)  610 AGGCACCCAGACTC lAslGslGsmdCsdAsmdCsmdCsmdCsdAsdGsdAslCslTslC (SEQ ID NO: 215) (SEQ ID NO: 216)  611 CAGGCACCCAGACT lCslAslGsdGsmdCsdAsmdCsmdCsmdCsdAsdGslAslCslT (SEQ ID NO: 217) (SEQ ID NO: 218)  612 TCAGGCACCCAGAC lTslCslAsdGsdGsmdCsdAsmdCsmdCsmdCsdAslGslAslC (SEQ ID NO: 219) (SEQ ID NO: 220)  613 ATCAGGCACCCAGA lAslTslCsdAsdGsdGsmdCsdAsmdCsmdCsmdCslAslGslA (SEQ ID NO: 221) (SEQ ID NO: 222) 1447 GCTGCAGAAGTGGT lGslCslTsdGsmdCsdAsdGsdAsdAsdGsdTslGslGslT (SEQ ID NO: 223) (SEQ ID NO: 224) 1518 ATGTCCATGGCATG lAslTslGsdTsmdCsmdCsdAsdTsdGsdGsmdCslAslTslG (SEQ ID NO: 225) (SEQ ID NO: 226) 1560 ATCTTATAGCAGGC lAslTslCsdTsdTsdAsdTsdAsdGsmdCsdAslGslGslC (SEQ ID NO: 227) (SEQ ID NO: 228) 1645 ACTCTCAGTGCCAT lAslCslTsmdCsdTsmdCsdAsdGsdTsdGsmdCslCslAslT (SEQ ID NO: 229) (SEQ ID NO: 230) 1646 CACTCTCAGTGCCA lCslAslCsdTsmdCsdTsmdCsdAsdGsdTsdGslCslCslA (SEQ ID NO: 231) (SEQ ID NO: 232) 1648 AGCACTCTCAGTGC lAslGslCsdAsmdCsdTsmdCsdTsmdCsdAsdGslTslGslC (SEQ ID NO: 233) (SEQ ID NO: 234) 1649 CAGCACTCTCAGTG lCslAslGsmdCsdAsmdCsdTsmdCsdTsmdCsdAslGslTslG (SEQ ID NO: 235) (SEQ ID NO: 236) 1650 TCAGCACTCTCAGT lTslCslAsdGsmdCsdAsmdCsdTsmdCsdTsmdCslAslGslT (SEQ ID NO: 237) (SEQ ID NO: 238) 1651 GTCAGCACTCTCAG lGslTslCsdAsdGsmdCsdAsmdCsdTsmdCsdTslCslAslG (SEQ ID NO: 239) (SEQ ID NO: 240) 1652 TGTCAGCACTCTCA lTslGslTsmdCsdAsdGsmdCsdAsmdCsdTsmdCslTslCslA (SEQ ID NO:241 ) (SEQ ID NO: 242) 1655 CCTTGTCAGCACTC lCslCslTsdTsdGsdTsmdCsdAsdGsmdCsdAslCslTslC (SEQ ID NO: 243) (SEQ ID NO: 244) 1682 CACTGCTGACCCCC lCslAslCsdTsdGsmdCsdTsdGsdAsmdCsmdCslCslCslC (SEQ ID NO: 245) (SEQ ID NO: 246) 1683 CCACTGCTGACCCC lCslCslAsmdCsdTsdGsmdCsdTsdGsdAsmdCslCslCslC (SEQ ID NO: 247) (SEQ ID NO: 248) 1684 CCCACTGCTGACCC lCslCslCsdAsmdCsdTsdGsmdCsdTsdGsdAslCslCslC (SEQ ID NO: 249) (SEQ ID NO: 250) 1685 CCCCACTGCTGACC lCslCslCsmdCsdAsmdCsdTsdGsmdCsdTsdGslAslCslC (SEQ ID NO: 251) (SEQ ID NO: 252) 1686 TCCCCACTGCTGAC lTslCslCsmdCsmdCsdAsmdCsdTsdGsmdCsdTslGslAslC (SEQ ID NO: 253) (SEQ ID NO: 254) 1687 GTCCCCACTGCTGA lGslTslCsmdCsmdCsmdCsdAsmdCsdTsdGsmdCslTslGslA (SEQ ID NO: 255) (SEQ ID NO: 256) 1688 GGTCCCCACTGCTG lGslGslTsmdCsmdCsmdCsmdCsdAsmdCsdTsdGslCslTslG (SEQ ID NO: 257) (SEQ ID NO: 258) 1689 AGGTCCCCACTGCT lAslGslGsdTsmdCsmdCsmdCsmdCsdAsmdCsdTslGslCslT (SEQ ID NO: 259) (SEQ ID NO: 260) 1690 GAGGTCCCCACTGC lGslAslGsdGsdTsmdCsmdCsmdCsmdCsdAsmdCslTslGslC (SEQ ID NO: 261) (SEQ ID NO: 262) 1691 GGAGGTCCCCACTG lGslGslAsdGsdGsdTsmdCsmdCsmdCsmdCsdAslCslTslG (SEQ ID NO: 263) (SEQ ID NO: 264) 1692 AGGAGGTCCCCACT lAslGslGsdAsdGsdGsdTsmdCsmdCsmdCsmdCslAslCslT (SEQ ID NO: 265) (SEQ ID NO: 266) 1693 GAGGAGGTCCCCAC lGslAslGsdGsdAsdGsdGsdTsmdCsmdCsmdCslCslAslC (SEQ ID NO: 267) (SEQ ID NO: 268) 1694 TGAGGAGGTCCCCA lTslGslAsdGsdGsdAsdGsdGsdTsmdCsmdCslCslCslA (SEQ ID NO: 269) (SEQ ID NO: 270) 1696 TTTGAGGAGGTCCC lTslTslTsdGsdAsdGsdGsdAsdGsdGsdTslCslCslC (SEQ ID NO: 271) (SEQ ID NO: 272) 1702 AAGGCCTTTGAGGA lAslAslGsdGsmdCsmdCsdTsdTsdTsdGsdAslGslGslA (SEQ ID NO: 273) (SEQ ID NO: 274) 1773 GCAAACACCACCTG lGslCslAsdAsdAsmdCsdAsmdCsmdCsdAsmdCslCslTslG (SEQ ID NO: 275) (SEQ ID NO: 276) 1774 AGCAAACACCACCT lAslGslCsdAsdAsdAsmdCsdAsmdCsmdCsdAslCslCslT (SEQ ID NO: 277) (SEQ ID NO: 278) 1775 CAGCAAACACCACC lCslAslGsmdCsdAsdAsdAsmdCsdAsmdCsmdCslAslCslC (SEQ ID NO:279 ) (SEQ ID NO: 280) 1776 ACAGCAAACACCAC lAslCslAsdGsmdCsdAsdAsdAsmdCsdAsmdCslCslAslC (SEQ ID NO: 281) (SEQ ID NO: 282) 1828 AGACACCAGCCACC lAslGslAsmdCsdAsmdCsmdCsdAsdGsmdCsmdCslAslCslC (SEQ ID NO: 283) (SEQ ID NO: 284) 1829 GAGACACCAGCCAC lGslAslGsdAsmdCsdAsmdCsmdCsdAsdGsmdCslCslAslC (SEQ ID NO: 285) (SEQ ID NO: 286) 2218 CTTCTTGTCAGGCC lCslTslTsmdCsdTsdTsdGsdTsmdCsdAsdGslGslCslC (SEQ ID NO: 287) (SEQ ID NO: 288) 2238 AAGTGGGCCTGGTA lAslAslGsdTsdGsdGsdGsmdCsmdCsdTsdGslGslTslA (SEQ ID NO: 289) (SEQ ID NO: 290) 2254 GTAGTGGACCACCT lGslTslAsdGsdTsdGsdGsdAsmdCsmdCsdAslCslCslT (SEQ ID NO: 291) (SEQ ID NO: 292) 2574 GCATCCATGACCCC lGslCslAsdTsmdCsmdCsdAsdTsdGsdAsmdCslCslCslC (SEQ ID NO: 293) (SEQ ID NO: 294) 2575 GGCATCCATGACCC lGslGslCsdAsdTsmdCsmdCsdAsdTsdGsdAslCslCslC (SEQ ID NO: 295) (SEQ ID NO: 296) 2576 AGGCATCCATGACC lAslGslGsmdCsdAsdTsmdCsmdCsdAsdTsdGslAslCslC (SEQ ID NO: 297) (SEQ ID NO: 298) 2577 AAGGCATCCATGAC lAslAslGsdGsmdCsdAsdTsmdCsmdCsdAsdTslGslAslC (SEQ ID NO: 299) (SEQ ID NO: 300) 4281 TGATCCTCATAGGT lTslGslAsdTsmdCsmdCsdTsmdCsdAsdTsdAslGslGslT (SEQ ID NO: 301) (SEQ ID NO: 302) 4282 CTGATCCTCATAGG lCslTslGsdAsdTsmdCsmdCsdTsmdCsdAsdTslAslGslG (SEQ ID NO: 303) (SEQ ID NO: 304) 4283 GCTGATCCTCATAG lGslCslTsdGsdAsdTsmdCsmdCsdTsmdCsdAslTslAslG (SEQ ID NO: 305) (SEQ ID NO: 306) 4284 AGCTGATCCTCATA lAslGslCsdTsdGsdAsdTsmdCsmdCsdTsmdCslAslTslA (SEQ ID NO: 307) (SEQ ID NO: 308) 4305 ACCTTGATCTTGGC lAslCslCsdTsdTsdGsdAsdTsmdCsdTsdTslGslGslC (SEQ ID NO: 309) (SEQ ID NO: 310) 4656 ATGGCATCTGCTTC lAslTslGsdGsmdCsdAsdTsmdCsdTsdGsmdCslTslTslC (SEQ ID NO: 311) (SEQ ID NO: 312) 4657 GATGGCATCTGCTT lGslAslTsdGsdGsmdCsdAsdTsmdCsdTsdGslCslTslT (SEQ ID NO: 313) (SEQ ID NO: 314) 4661 TCTGGATGGCATCT lTslCslTsdGsdGsdAsdTsdGsdGsmdCsdAslTslCslT (SEQ ID NO: 315) (SEQ ID NO: 316) 6141 ACCTTGCTCTGCAG lAslCslCsdTsdTsdGsmdCsdTsmdCsdTsdGslCslAslG (SEQ ID NO: 317) (SEQ ID NO: 318) 436 AGGCAGCAGGCACT lAslGslGsmdCsdAsdGsmdCsdAsdGsdGsmdCslAslCslT (SEQ ID NO: 319) (SEQ ID NO: 320) 437 AAGGCAGCAGGCAC lAslAslGsdGsmdCsdAsdGsmdCsdAsdGsdGslCslAslC (SEQ ID NO: 321) (SEQ ID NO: 322) 438 CAAGGCAGCAGGCA lCslAslAsdGsdGsmdCsdAsdGsmdCsdAsdGslGslCslA (SEQ ID NO: 323) (SEQ ID NO: 324) 439 CCAAGGCAGCAGGC lCslCslAsdAsdGsdGsmdCsdAsdGsmdCsdAslGslGslC (SEQ ID NO: 325) (SEQ ID NO: 326) 482 GGTGAAGGAGGAGG lGslGslTsdGsdAsdAsdGsdGsdAsdGsdGslAslGslG (SEQ ID NO: 327) (SEQ ID NO: 328) 483 GGGTGAAGGAGGAG lGslGslGsdTsdGsdAsdAsdGsdGsdAsdGslGslAslG (SEQ ID NO: 329) (SEQ ID NO: 330) 509 TCACATCCATCATG lTslCslAsmdCsdAsdTsmdCsmdCsdAsdTsmdCslAslTslG (SEQ ID NO: 331) (SEQ ID NO: 332) 511 ACTCACATCCATCA lAslCslTsmdCsdAsmdCsdAsdTsmdCsmdCsdAslTslCslA (SEQ ID NO: 333) (SEQ ID NO: 334) 512 CACTCACATCCATC lCslAslCsdTsmdCsdAsmdCsdAsdTsmdCsmdCslAslTslC (SEQ ID NO: 335) (SEQ ID NO: 336) 616 TTCATCAGGCACCC lTslTslCsdAsdTsmdCsdAsdGsdGsmdCsdAslCslCslC (SEQ ID NO: 337) (SEQ ID NO: 338) 617 GTTCATCAGGCACC lGslTslTsmdCsdAsdTsmdCsdAsdGsdGsmdCslAslCslC (SEQ ID NO: 339) (SEQ ID NO: 340) 622 GTCCTGTTCATCAG lGslTslCsmdCsdTsdGsdTsdTsmdCsdAsdTslCslAslG (SEQ ID NO: 341) (SEQ ID NO: 342) 690 TGGTCTTTGGTCTC lTslGslGsdTsmdCsdTsdTsdTsdGsdGsdTslCslTslC (SEQ ID NO: 343) (SEQ ID NO: 344) 804 TTGTGCAGCACAGA lTslTslGsdTsdGsmdCsdAsdGsmdCsdAsmdCslAslGslA (SEQ ID NO: 345) (SEQ ID NO: 346) 805 GTTGTGCAGCACAG lGslTslTsdGsdTsdGsmdCsdAsdGsmdCsdAslCslAslG (SEQ ID NO: 347) (SEQ ID NO: 348) 892 GACTGGGAGCCATT lGslAslCsdTsdGsdGsdGsdAsdGsmdCsmdCslAslTslT (SEQ ID NO: 349) (SEQ ID NO: 350) 893 AGACTGGGAGCCAT lAslGslAsmdCsdTsdGsdGsdGsdAsdGsmdCslCslAslT (SEQ ID NO: 351) (SEQ ID NO: 352) 1102 GTCTCCCAGGGCAG lGslTslCsdTsmdCsmdCsmdCsdAsdGsdGsdGslCslAslG (SEQ ID NO: 353) (SEQ ID NO: 354) 1215 CTCAGGGTCTTGGC lCslTslCsdAsdGsdGsdGsdTsmdCsdTsdTslGslGslC (SEQ ID NO: 355) (SEQ ID NO: 356) 1216 CCTCAGGGTCTTGG lCslCslTsmdCsdAsdGsdGsdGsdTsmdCsdTslTslGslG (SEQ ID NO: 357) (SEQ ID NO: 358) 1217 TCCTCAGGGTCTTG lTslCslCsdTsmdCsdAsdGsdGsdGsdTsmdCslTslTslG (SEQ ID NO: 359) (SEQ ID NO: 360) 1219 ATTCCTCAGGGTCT lAslTslTsmdCsmdCsdTsmdCsdAsdGsdGsdGslTslCslT (SEQ ID NO: 361) (SEQ ID NO: 362) 1220 CATTCCTCAGGGTC lCslAslTsdTsmdCsmdCsdTsmdCsdAsdGsdGslGslTslC (SEQ ID NO: 363) (SEQ ID NO: 364) 1222 ATCATTCCTCAGGG lAslTslCsdAsdTsdTsmdCsmdCsdTsmdCsdAslGslGslG (SEQ ID NO: 365) (SEQ ID NO: 366) 1225 GTTATCATTCCTCA lGslTslTsdAsdTsmdCsdAsdTsdTsmdCsmdCslTslCslA (SEQ ID NO: 366) (SEQ ID NO: 368) 1226 AGTTATCATTCCTC lAslGslTsdTsdAsdTsmdCsdAsdTsdTsmdCslCslTslC (SEQ ID NO: 369) (SEQ ID NO: 370) 1268 AGGGACCAAAGTGA lAslGslGsdGsdAsmdCsmdCsdAsdAsdAsdGslTslGslA (SEQ ID NO: 371) (SEQ ID NO: 372) 1273 CCCAGAGGGACCAA lCslCslCsdAsdGsdAsdGsdGsdGsdAsmdCslCslAslA (SEQ ID NO: 373) (SEQ ID NO: 374) 1274 TCCCAGAGGGACCA lTslCslCsmdCsdAsdGsdAsdGsdGsdGsdAslCslCslA (SEQ ID NO: 375) (SEQ ID NO: 376) 1275 TTCCCAGAGGGACC lTslTslCsmdCsmdCsdAsdGsdAsdGsdGsdGslAslCslC (SEQ ID NO: 377) (SEQ ID NO: 378) 1276 CTTCCCAGAGGGAC lCslTslTsmdCsmdCsmdCsdAsdGsdAsdGsdGslGslAslC (SEQ ID NO: 379) (SEQ ID NO: 380) 1277 GCTTCCCAGAGGGA lGslCslTsdTsmdCsmdCsmdCsdAsdGsdAsdGslGslGslA (SEQ ID NO: 381) (SEQ ID NO: 382) 1301 AGCTGTCAATATCC lAslGslCsdTsdGsdTsmdCsdAsdAsdTsdAslTslCslC (SEQ ID NO: 383) (SEQ ID NO: 384) 1302 TAGCTGTCAATATC lTslAslGsmdCsdTsdGsdTsmdCsdAsdAsdTslAslTslC (SEQ ID NO: 385) (SEQ ID NO: 386) 1303 ATAGCTGTCAATAT lAslTslAsdGsmdCsdTsdGsdTsmdCsdAsdAslTslAslT (SEQ ID NO: 387) (SEQ ID NO: 388) 1304 GATAGCTGTCAATA lGslAslTsdAsdGsmdCsdTsdGsdTsmdCsdAslAslTslA (SEQ ID NO: 389) (SEQ ID NO: 390) 1379 CTGAGAGGATCTGG lCslTslGsdAsdGsdAsdGsdGsdAsdTsmdCslTslGslG (SEQ ID NO: 391) (SEQ ID NO: 392) 1380 CCTGAGAGGATCTG lCslCslTsdGsdAsdGsdAsdGsdGsdAsdTslCslTslG (SEQ ID NO: 393) (SEQ ID NO: 394) 1417 AGACAGAAGCAGCA lAslGslAsmdCsdAsdGsdAsdAsdGsmdCsdAslGslCslA (SEQ ID NO: 395) (SEQ ID NO: 396) 1418 TAGACAGAAGCAGC lTslAslGsdAsmdCsdAsdGsdAsdAsdGsmdCslAslGslC (SEQ ID NO: 397) (SEQ ID NO: 398) 1419 ATAGACAGAAGCAG lAslTslAsdGsdAsmdCsdAsdGsdAsdAsdGslCslAslG (SEQ ID NO: 399) (SEQ ID NO: 400) 1420 CATAGACAGAAGCA lCslAslTsdAsdGsdAsmdCsdAsdGsdAsdAslGslCslA (SEQ ID NO: 401) (SEQ ID NO: 402) 1421 TCATAGACAGAAGC lTslCslAsdTsdAsdGsdAsmdCsdAsdGsdAslAslGslC (SEQ ID NO: 403) (SEQ ID NO: 404) 1422 TTCATAGACAGAAG lTslTslCsdAsdTsdAsdGsdAsmdCsdAsdGslAslAslG (SEQ ID NO: 405) (SEQ ID NO: 406) 1423 GTTCATAGACAGAA lGslTslTsmdCsdAsdTsdAsdGsdAsmdCsdAslGslAslA (SEQ ID NO: 407) (SEQ ID NO: 408) 1424 GGTTCATAGACAGA lGslGslTsdTsmdCsdAsdTsdAsdGsdAsmdCslAslGslA (SEQ ID NO: 409) (SEQ ID NO: 410) 1425 GGGTTCATAGACAG lGslGslGsdTsdTsmdCsdAsdTsdAsdGsdAslCslAslG (SEQ ID NO: 411) (SEQ ID NO: 412) 1484 CCCCATCATTCATG lCslCslCsmdCsdAsdTsmdCsdAsdTsdTsmdCslAslTslG (SEQ ID NO: 413) (SEQ ID NO: 414) 1557 TTATAGCAGGCACA lTslTslAsdTsdAsdGsmdCsdAsdGsdGsmdCslAslCslA (SEQ ID NO: 415) (SEQ ID NO: 416) 1558 CTTATAGCAGGCAC lCslTslTsdAsdTsdAsdGsmdCsdAsdGsdGslCslAslC (SEQ ID NO: 417) (SEQ ID NO: 418) 1559 TCTTATAGCAGGCA lTslCslTsdTsdAsdTsdAsdGsmdCsdAsdGslGslCslA (SEQ ID NO: 419) (SEQ ID NO: 420) 1561 GATCTTATAGCAGG lGslAslTsmdCsdTsdTsdAsdTsdAsdGsmdCslAslGslG (SEQ ID NO: 421) (SEQ ID NO: 422) 1582 AAAGTGCAGGAGGG lAslAslAsdGsdTsdGsmdCsdAsdGsdGsdAslGslGslG (SEQ ID NO: 423) (SEQ ID NO: 424) 1587 TTGCCAAAGTGCAG lTslTslGsmdCsmdCsdAsdAsdAsdGsdTsdGslCslAslG (SEQ ID NO: 425) (SEQ ID NO: 426) 1703 AAAGGCCTTTGAGG lAslAslAsdGsdGsmdCsmdCsdTsdTsdTsdGslAslGslG (SEQ ID NO: 427) (SEQ ID NO: 428) 1704 AAAAGGCCTTTGAG lAslAslAsdAsdGsdGsmdCsmdCsdTsdTsdTslGslAslG (SEQ ID NO: 429) (SEQ ID NO: 430) 1705 CAAAAGGCCTTTGA lCslAslAsdAsdAsdGsdGsmdCsmdCsdTsdTslTslGslA (SEQ ID NO: 431) (SEQ ID NO: 432) 1706 GCAAAAGGCCTTTG lGslCslAsdAsdAsdAsdGsdGsmdCsmdCsdTslTslTslG (SEQ ID NO: 433) (SEQ ID NO: 434) 1707 TGCAAAAGGCCTTT lTslGslCsdAsdAsdAsdAsdGsdGsmdCsmdCslTslTslT (SEQ ID NO: 435) (SEQ ID NO: 436) 1708 GTGCAAAAGGCCTT lGslTslGsmdCsdAsdAsdAsdAsdGsdGsmdCslCslTslT (SEQ ID NO: 437) (SEQ ID NO: 438) 1709 GGTGCAAAAGGCCT lGslGslTsdGsmdCsdAsdAsdAsdAsdGsdGslCslCslT (SEQ ID NO: 439) (SEQ ID NO: 440) 1710 GGGTGCAAAAGGCC lGslGslGsdTsdGsmdCsdAsdAsdAsdAsdGslGslCslC (SEQ ID NO: 441) (SEQ ID NO: 442) 1804 GTCATAGGTGGCCT lGslTslCsdAsdTsdAsdGsdGsdTsdGsdGslCslCslT (SEQ ID NO: 443) (SEQ ID NO: 444) 1805 GGTCATAGGTGGCC lGslGslTsmdCsdAsdTsdAsdGsdGsdTsdGslGslCslC (SEQ ID NO: 445) (SEQ ID NO: 446) 1819 CCACCTGAACAGCC lCslCslAsmdCsmdCsdTsdGsdAsdAsmdCsdAslGslCslC (SEQ ID NO: 447) (SEQ ID NO: 448) 1820 GCCACCTGAACAGC lGslCslCsdAsmdCsmdCsdTsdGsdAsdAsmdCslAslGslC (SEQ ID NO: 449) (SEQ ID NO: 450) 1827 GACACCAGCCACCT lGslAslCsdAsmdCsmdCsdAsdGsmdCsmdCsdAslCslCslT (SEQ ID NO: 451) (SEQ ID NO: 452) 1850 TGTCCAGGGTCTGG lTslGslTsmdCsmdCsdAsdGsdGsdGsdTsmdCslTslGslG (SEQ ID NO: 453) (SEQ ID NO: 454) 1851 GTGTCCAGGGTCTG lGslTslGsdTsmdCsmdCsdAsdGsdGsdGsdTslCslTslG (SEQ ID NO: 455) (SEQ ID NO: 456) 1852 TGTGTCCAGGGTCT lTslGslTsdGsdTsmdCsmdCsdAsdGsdGsdGslTslCslT (SEQ ID NO: 457) (SEQ ID NO: 458) 2189 TGGGTGACTTCCCC lTslGslGsdGsdTsdGsdAsmdCsdTsdTsmdCslCslCslC (SEQ ID NO: 459) (SEQ ID NO: 460) 2190 TTGGGTGACTTCCC lTslTslGsdGsdGsdTsdGsdAsmdCsdTsdTslCslCslC (SEQ ID NO: 461) (SEQ ID NO: 462) 2191 ATTGGGTGACTTCC lAslTslTsdGsdGsdGsdTsdGsdAsmdCsdTslTslCslC (SEQ ID NO: 463) (SEQ ID NO: 464) 2198 GCTGGAAATTGGGT lGslCslTsdGsdGsdAsdAsdAsdTsdTsdGslGslGslT (SEQ ID NO: 465) (SEQ ID NO: 466) 2274 CTGTAAGGCACCAC lCslTslGsdTsdAsdAsdGsdGsmdCsdAsmdCslCslAslC (SEQ ID NO: 467) (SEQ ID NO: 468) 2275 GCTGTAAGGCACCA lGslCslTsdGsdTsdAsdAsdGsdGsmdCsdAslCslCslA (SEQ ID NO: 469) (SEQ ID NO: 470) 2276 TGCTGTAAGGCACC lTslGslCsdTsdGsdTsdAsdAsdGsdGsmdCslAslCslC (SEQ ID NO: 471) (SEQ ID NO: 472) 2350 ATTCTGTGACTTCT lAslTslTsmdCsdTsdGsdTsdGsdAsmdCsdTslTslCslT (SEQ ID NO: 473) (SEQ ID NO: 474) 2351 TATTCTGTGACTTC lTslAslTsdTsmdCsdTsdGsdTsdGsdAsmdCslTslTslC (SEQ ID NO: 475) (SEQ ID NO: 476) 2352 CTATTCTGTGACTT lCslTslAsdTsdTsmdCsdTsdGsdTsdGsdAslCslTslT (SEQ ID NO: 477) (SEQ ID NO: 478) 2353 CCTATTCTGTGACT lCslCslTsdAsdTsdTsmdCsdTsdGsdTsdGslAslCslT (SEQ ID NO: 479) (SEQ ID NO: 480) 2354 GCCTATTCTGTGAC lGslCslCsdTsdAsdTsdTsmdCsdTsdGsdTslGslAslC (SEQ ID NO: 481) (SEQ ID NO: 482) 2355 AGCCTATTCTGTGA lAslGslCsmdCsdTsdAsdTsdTsmdCsdTsdGslTslGslA (SEQ ID NO: 483) (SEQ ID NO: 484) 2356 GAGCCTATTCTGTG lGslAslGsmdCsmdCsdTsdAsdTsdTsmdCsdTslGslTslG (SEQ ID NO: 485) (SEQ ID NO: 486) 2357 GGAGCCTATTCTGT lGslGslAsdGsmdCsmdCsdTsdAsdTsdTsmdCslTslGslT (SEQ ID NO: 487) (SEQ ID NO: 488) 2358 AGGAGCCTATTCTG lAslGslGsdAsdGsmdCsmdCsdTsdAsdTsdTslCslTslG (SEQ ID NO: 489) (SEQ ID NO: 490) 2359 CAGGAGCCTATTCT lCslAslGsdGsdAsdGsmdCsmdCsdTsdAsdTslTslCslT (SEQ ID NO: 491) (SEQ ID NO: 492) 2360 CCAGGAGCCTATTC lCslCslAsdGsdGsdAsdGsmdCsmdCsdTsdAslTslTslC (SEQ ID NO: 493) (SEQ ID NO: 494) 2361 GCCAGGAGCCTATT lGslCslCsdAsdGsdGsdAsdGsmdCsmdCsdTslAslTslT (SEQ ID NO: 495) (SEQ ID NO: 496) 2375 TCTCATAGAGAGTC lTslCslTsmdCsdAsdTsdAsdGsdAsdGsdAslGslTslC (SEQ ID NO: 497) (SEQ ID NO: 498) 2376 TTCTCATAGAGAGT lTslTslCsdTsmdCsdAsdTsdAsdGsdAsdGslAslGslT (SEQ ID NO: 499) (SEQ ID NO: 500) 2396 TGGAGCAGGAGCCC lTslGslGsdAsdGsmdCsdAsdGsdGsdAsdGslCslCslC (SEQ ID NO: 501) (SEQ ID NO: 502) 2397 GTGGAGCAGGAGCC lGslTslGsdGsdAsdGsmdCsdAsdGsdGsdAslGslCslC (SEQ ID NO: 503) (SEQ ID NO: 504) 2443 TGCTGCCTTCTTAC lTslGslCsdTsdGsmdCsmdCsdTsdTsmdCsdTslTslAslC (SEQ ID NO: 505) (SEQ ID NO: 506) 2444 ATGCTGCCTTCTTA lAslTslGsmdCsdTsdGsmdCsmdCsdTsdTsmdCslTslTslA (SEQ ID NO: 507) (SEQ ID NO: 508) 2445 GATGCTGCCTTCTT lGslAslTsdGsmdCsdTsdGsmdCsmdCsdTsdTslCslTslT (SEQ ID NO: 509) (SEQ ID NO: 510) 2579 AGAAGGCATCCATG lAslGslAsdAsdGsdGsmdCsdAsdTsmdCsmdCslAslTslG (SEQ ID NO: 511) (SEQ ID NO: 512) 2580 AAGAAGGCATCCAT lAslAslGsdAsdAsdGsdGsmdCsdAsdTsmdCslCslAslT (SEQ ID NO: 513) (SEQ ID NO: 514) 2581 CAAGAAGGCATCCA lCslAslAsdGsdAsdAsdGsdGsmdCsdAsdTslCslCslA (SEQ ID NO: 515) (SEQ ID NO: 516) 2584 CACCAAGAAGGCAT lCslAslCsmdCsdAsdAsdGsdAsdAsdGsdGslCslAslT (SEQ ID NO: 517) (SEQ ID NO: 518) 2585 GCACCAAGAAGGCA lGslCslAsmdCsmdCsdAsdAsdGsdAsdAsdGslGslCslA (SEQ ID NO: 519) (SEQ ID NO: 520) 2587 TAGCACCAAGAAGG lTslAslGsmdCsdAsmdCsmdCsdAsdAsdGsdAslAslGslG (SEQ ID NO: 521) (SEQ ID NO: 522) 2588 GTAGCACCAAGAAG lGslTslAsdGsmdCsdAsmdCsmdCsdAsdAsdGslAslAslG (SEQ ID NO: 523) (SEQ ID NO: 524) 2589 TGTAGCACCAAGAA lTslGslTsdAsdGsmdCsdAsmdCsmdCsdAsdAslGslAslA (SEQ ID NO: 525) (SEQ ID NO: 526) 2590 GTGTAGCACCAAGA lGslTslGsdTsdAsdGsmdCsdAsmdCsmdCsdAslAslGslA (SEQ ID NO: 527) (SEQ ID NO: 528) 2591 GGTGTAGCACCAAG lGslGslTsdGsdTsdAsdGsmdCsdAsmdCsmdCslAslAslG (SEQ ID NO: 529) (SEQ ID NO: 530) 2592 TGGTGTAGCACCAA lTslGslGsdTsdGsdTsdAsdGsmdCsdAsmdCslCslAslA (SEQ ID NO: 531) (SEQ ID NO: 532) 2709 GGGATGGCACTGGG lGslGslGsdAsdTsdGsdGsmdCsdAsmdCsdTslGslGslG (SEQ ID NO: 533) (SEQ ID NO: 534) 2731 GCTGTCCATGAAGG lGslCslTsdGsdTsmdCsmdCsdAsdTsdGsdAslAslGslG (SEQ ID NO: 535) (SEQ ID NO: 536) 2830 GCCTAGAAGCCCAG lGslCslCsdTsdAsdGsdAsdAsdGsmdCsmdCslCslAslG (SEQ ID NO: 537) (SEQ ID NO: 538) 2986 GGCATTGAAGGCAC lGslGslCsdAsdTsdTsdGsdAsdAsdGsdGslCslAslC (SEQ ID NO: 539) (SEQ ID NO: 540) 3248 ACTTGATCAGCAAG lAslCslTsdTsdGsdAsdTsmdCsdAsdGsmdCslAslAslG (SEQ ID NO: 541) (SEQ ID NO: 542) 3249 GACTTGATCAGCAA lGslAslCsdTsdTsdGsdAsdTsmdCsdAsdGslCslAslA (SEQ ID NO: 543) (SEQ ID NO: 544) 3625 GCATTCCAGGTCCT lGslCslAsdTsdTsmdCsmdCsdAsdGsdGsdTslCslCslT (SEQ ID NO: 545) (SEQ ID NO: 546) 3626 AGCATTCCAGGTCC lAslGslCsdAsdTsdTsmdCsmdCsdAsdGsdGslTslCslC (SEQ ID NO: 547) (SEQ ID NO: 548) 3628 GGAGCATTCCAGGT lGslGslAsdGsmdCsdAsdTsdTsmdCsmdCsdAslGslGslT (SEQ ID NO: 549) (SEQ ID NO: 550) 3629 GGGAGCATTCCAGG lGslGslGsdAsdGsmdCsdAsdTsdTsmdCsmdCslAslGslG (SEQ ID NO: 551) (SEQ ID NO: 552) 3631 CAGGGAGCATTCCA lCslAslGsdGsdGsdAsdGsmdCsdAsdTsdTslCslCslA (SEQ ID NO: 553) (SEQ ID NO: 554) 3793 CAGGCTCAGCTGGC lCslAslGsdGsmdCsdTsmdCsdAsdGsmdCsdTslGslGslC (SEQ ID NO: 555) (SEQ ID NO: 556) 3794 GCAGGCTCAGCTGG lGslCslAsdGsdGsmdCsdTsmdCsdAsdGsmdCslTslGslG (SEQ ID NO: 557) (SEQ ID NO: 558) 4233 GTCAGAGTCTCCAC lGslTslCsdAsdGsdAsdGsdTsmdCsdTsmdCslCslAslC (SEQ ID NO: 559) (SEQ ID NO: 560) 4234 GGTCAGAGTCTCCA lGslGslTsmdCsdAsdGsdAsdGsdTsmdCsdTslCslCslA (SEQ ID NO: 561) (SEQ ID NO: 562) 4235 GGGTCAGAGTCTCC lGslGslGsdTsmdCsdAsdGsdAsdGsdTsmdCslTslCslC (SEQ ID NO: 563) (SEQ ID NO: 564) 4286 TTAGCTGATCCTCA lTslTslAsdGsmdCsdTsdGsdAsdTsmdCsmdCslTslCslA (SEQ ID NO: 565) (SEQ ID NO: 566) 4287 CTTAGCTGATCCTC lCslTslTsdAsdGsmdCsdTsdGsdAsdTsmdCslCslTslC (SEQ ID NO: 567) (SEQ ID NO: 568) 4410 ATCAGACACTCCTT lAslTslCsdAsdGsdAsmdCsdAsmdCsdTsmdCslCslTslT (SEQ ID NO: 569) (SEQ ID NO: 570) 4412 TGATCAGACACTCC lTslGslAsdTsmdCsdAsdGsdAsmdCsdAsmdCslTslCslC (SEQ ID NO: 571) (SEQ ID NO: 572) 4413 CTGATCAGACACTC lCslTslGsdAsdTsmdCsdAsdGsdAsmdCsdAslCslTslC (SEQ ID NO: 573) (SEQ ID NO: 574) 4414 ACTGATCAGACACT lAslCslTsdGsdAsdTsmdCsdAsdGsdAsmdCslAslCslT (SEQ ID NO: 575) (SEQ ID NO: 576) 4415 GACTGATCAGACAC lGslAslCsdTsdGsdAsdTsmdCsdAsdGsdAslCslAslC (SEQ ID NO: 577) (SEQ ID NO: 578) 4416 TGACTGATCAGACA lTslGslAsmdCsdTsdGsdAsdTsmdCsdAsdGslAslCslA (SEQ ID NO: 579) (SEQ ID NO: 580) 4417 CTGACTGATCAGAC lCslTslGsdAsmdCsdTsdGsdAsdTsmdCsdAslGslAslC (SEQ ID NO: 581) (SEQ ID NO: 582) 4418 GCTGACTGATCAGA lGslCslTsdGsdAsmdCsdTsdGsdAsdTsmdCslAslGslA (SEQ ID NO: 583) (SEQ ID NO: 584) 4419 AGCTGACTGATCAG lAslGslCsdTsdGsdAsmdCsdTsdGsdAsdTslCslAslG (SEQ ID NO: 585) (SEQ ID NO: 586) 4422 CTCAGCTGACTGAT lCslTslCsdAsdGsmdCsdTsdGsdAsmdCsdTslGslAslT (SEQ ID NO: 587) (SEQ ID NO: 588) 4423 GCTCAGCTGACTGA lGslCslTsmdCsdAsdGsmdCsdTsdGsdAsmdCslTslGslA (SEQ ID NO: 589) (SEQ ID NO: 590) 4424 GGCTCAGCTGACTG lGslGslCsdTsmdCsdAsdGsmdCsdTsdGsdAslCslTslG (SEQ ID NO: 591) (SEQ ID NO: 592) 4797 TCCTCTGACTCTGT lTslCslCsdTsmdCsdTsdGsdAsmdCsdTsmdCslTslGslT (SEQ ID NO: 593) (SEQ ID NO: 594) 4798 ATCCTCTGACTCTG lAslTslCsmdCsdTsmdCsdTsdGsdAsmdCsdTslCslTslG (SEQ ID NO: 595) (SEQ ID NO: 596) 4799 CATCCTCTGACTCT lCslAslTsmdCsmdCsdTsmdCsdTsdGsdAsmdCslTslCslT (SEQ ID NO: 597) (SEQ ID NO: 598) 4800 ACATCCTCTGACTC lAslCslAsdTsmdCsmdCsdTsmdCsdTsdGsdAslCslTslC (SEQ ID NO: 599) (SEQ ID NO: 600) 4801 TACATCCTCTGACT lTslAslCsdAsdTsmdCsmdCsdTsmdCsdTsdGslAslCslT (SEQ ID NO: 601) (SEQ ID NO: 602) 4802 TTACATCCTCTGAC lTslTslAsmdCsdAsdTsmdCsmdCsdTsmdCsdTslGslAslC (SEQ ID NO: 603) (SEQ ID NO: 604) 4803 GTTACATCCTCTGA lGslTslTsdAsmdCsdAsdTsmdCsmdCsdTsmdCslTslGslA (SEQ ID NO: 605) (SEQ ID NO: 606) 4804 GGTTACATCCTCTG lGslGslTsdTsdAsmdCsdAsdTsmdCsmdCsdTslCslTslG (SEQ ID NO: 607) (SEQ ID NO: 608) 4805 GGGTTACATCCTCT lGslGslGsdTsdTsdAsmdCsdAsdTsmdCsmdCslTslCslT (SEQ ID NO: 609) (SEQ ID NO: 610) 4806 AGGGTTACATCCTC lAslGslGsdGsdTsdTsdAsmdCsdAsdTsmdCslCslTslC (SEQ ID NO: 611) (SEQ ID NO: 612) 4807 CAGGGTTACATCCT lCslAslGsdGsdGsdTsdTsdAsmdCsdAsdTslCslCslT (SEQ ID NO: 613) (SEQ ID NO: 614) 4808 CCAGGGTTACATCC lCslCslAsdGsdGsdGsdTsdTsdAsmdCsdAslTslCslC (SEQ ID NO: 615) (SEQ ID NO: 616) 4811 GCTCCAGGGTTACA lGslCslTsmdCsmdCsdAsdGsdGsdGsdTsdTslAslCslA (SEQ ID NO: 617) (SEQ ID NO: 618) 4812 AGCTCCAGGGTTAC lAslGslCsdTsmdCsmdCsdAsdGsdGsdGsdTslTslAslC (SEQ ID NO: 619) (SEQ ID NO: 620) 4836 GCAGCTGCTGAGGT lGslCslAsdGsmdCsdTsdGsmdCsdTsdGsdAslGslGslT (SEQ ID NO: 621) (SEQ ID NO: 622) 4837 AGCAGCTGCTGAGG lAslGslCsdAsdGsmdCsdTsdGsmdCsdTsdGslAslGslG (SEQ ID NO: 623) (SEQ ID NO: 624) 6033 CCCTTAAGGGCCTC lCslCslCsdTsdTsdAsdAsdGsdGsdGsmdCslCslTslC (SEQ ID NO: 625) (SEQ ID NO: 626) l = locked nucleotide; d = deoxynucleotide; mdC = 5-Methyl cytosine; m = 2′-O-methyl nucleotide; s = phosphorothioate linkage

TABLE 3 Starting nucleotide of target sequence in SEQ ID NO: 6 Sequence (5′-3′) Sequence (5′-3′) with modifications  436 AGGCAGCAGGCACT lAsdGslGsdCslAslGsdCsdAslGsdGslCsdAslCslT (SEQ ID NO: 627) (SEQ ID NO: 628)  513 TCACTCACATCCAT lTsdCslAsdCslTslCsdAsdCslAsdTslCsdCslAslT (SEQ ID NO: 629) (SEQ ID NO: 630)  524 ACTCCCCAAGTTCA lAsdCslTsdCslCslCsdCsdAslAsdGslTsdTslCslA (SEQ ID NO: 631) (SEQ ID NO: 632)  610 AGGCACCCAGACTC lAsdGslGsdCslAslCsdCsdCslAsdGslAsdCslTslC (SEQ ID NO: 633) (SEQ ID NO: 634)  735 GGGTTCATGGGCTG lGsdGslGsdTslTslCsdAsdTslGsdGslGsdCslTslG (SEQ ID NO: 635) (SEQ ID NO: 636)  867 ATGGTGACACAGAA lAsdTslGsdGslTslGsdAsdCslAsdCslAsdGslAslA (SEQ ID NO: 637) (SEQ ID NO: 638) 1020 ATCAGCATGGACTG lAsdTslCsdAslGslCsdAsdTslGsdGslAsdCslTslG (SEQ ID NO: 639) (SEQ ID NO: 640) 1061 GCTTGGTGTTAACC lGsdCslTsdTslGslGsdTsdGslTsdTslAsdAslCslC (SEQ ID NO: 641) (SEQ ID NO: 642) 1188 TCCATGGCAGGGTT lTsdCslCsdAslTslGsdGsdCslAsdGslGsdGslTslT (SEQ ID NO: 643) (SEQ ID NO: 644) 1305 AGATAGCTGTCAAT lAsdGslAsdTslAslGsdCsdTslGsdTslCsdAslAslT (SEQ ID NO: 645) (SEQ ID NO: 646) 1443 CAGAAGTGGTAGTC lCsdAslGsdAslAslGsdTsdGslGsdTslAsdGslTslC (SEQ ID NO: 647) (SEQ ID NO: 648) 1447 GCTGCAGAAGTGGT lGsdCslTsdGslCslAsdGsdAslAsdGslTsdGslGslT (SEQ ID NO: 649) (SEQ ID NO: 650) 1518 ATGTCCATGGCATG lAsdTslGsdTslCslCsdAsdTslGsdGslCsdAslTslG (SEQ ID NO: 651) (SEQ ID NO: 652) 1560 ATCTTATAGCAGGC lAsdTslCsdTslTslAsdTsdAslGsdCslAsdGslGslC (SEQ ID NO: 653) (SEQ ID NO: 654) 1647 GCACTCTCAGTGCC lGsdCslAsdCslTslCsdTsdCslAsdGslTsdGslCslC (SEQ ID NO: 655) (SEQ ID NO: 656) 1662 TAGGCAGCCTTGTC lTsdAslGsdGslCslAsdGsdCslCsdTslTsdGslTslC (SEQ ID NO: 657) (SEQ ID NO: 658) 1694 TGAGGAGGTCCCCA lTsdGslAsdGslGslAsdGsdGslTsdCslCsdCslCslA (SEQ ID NO: 659) (SEQ ID NO: 660) 1755 ACACTCTGGCCCTT lAsdCslAsdCslTslCsdTsdGslGsdCslCsdCslTslT (SEQ ID NO: 661) (SEQ ID NO: 662) 1764 ACCTGCTCCACACT lAsdCslCsdTslGslCsdTsdCslCsdAslCsdAslCslT (SEQ ID NO: 663) (SEQ ID NO: 664) 1828 AGACACCAGCCACC lAsdGslAsdCslAslCsdCsdAslGsdCslCsdAslCslC (SEQ ID NO: 665) (SEQ ID NO: 666) 1912 CTCAAAGATCTCAA lCsdTslCsdAslAslAsdGsdAslTsdCslTsdCslAslA (SEQ ID NO: 667) (SEQ ID NO: 668) 2037 ATGAAGACCCAGTC lAsdTslGsdAslAslGsdAsdCslCsdCslAsdGslTslC (SEQ ID NO: 669) (SEQ ID NO: 670) 2218 CTTCTTGTCAGGCC lCsdTslTsdCslTslTsdGsdTslCsdAslGsdGslCslC (SEQ ID NO: 671) (SEQ ID NO: 672) 2253 TAGTGGACCACCTC lTsdAslGsdTslGslGsdAsdCslCsdAslCsdCslTslC (SEQ ID NO: 673) (SEQ ID NO: 674) 2481 AGGTTCTCCTTGTG lAsdGslGsdTslTslCsdTsdCslCsdTslTsdGslTslG (SEQ ID NO: 675) (SEQ ID NO: 676) 2518 GGGCTGTGTGGCCC lGsdGslGsdCslTslGsdTsdGslTsdGslGsdCslCslC (SEQ ID NO: 677) (SEQ ID NO: 678) 2738 CCTTCCTGCTGTCC lCsdCslTsdTslCslCsdTsdGslCsdTslGsdTslCslC (SEQ ID NO: 679) (SEQ ID NO: 680) 2961 TGGATGGTGAACAG lTsdGslGsdAslTslGsdGsdTslGsdAslAsdCslAslG (SEQ ID NO: 681) (SEQ ID NO: 682) 3186 TGCAGGGCCAGGTC lTsdGslCsdAslGslGsdGsdCslCsdAslGsdGslTslC (SEQ ID NO: 683) (SEQ ID NO: 684) 3255 ACCTTGGACTTGAT lAsdCslCsdTslTslGsdGsdAslCsdTslTsdGslAslT (SEQ ID NO: 685) (SEQ ID NO: 686) 3625 GCATTCCAGGTCCT lGsdCslAsdTslTslCsdCsdAslGsdGslTsdCslCslT (SEQ ID NO: 687) (SEQ ID NO: 688) 3794 GCAGGCTCAGCTGG lGsdCslAsdGslGslCsdTsdCslAsdGslCsdTslGslG (SEQ ID NO: 689) (SEQ ID NO: 690) 4253 CTGCACTGGCCTTG lCsdTslGsdCslAslCsdTsdGslGsdCslCsdTslTslG (SEQ ID NO: 691) (SEQ ID NO: 692) 4259 GCTTCTCTGCACTG lGsdCslTsdTslCslTsdCsdTslGsdCslAsdCslTslG (SEQ ID NO: 693) (SEQ ID NO: 694) 4302 TTGATCTTGGCCTC lTsdTslGsdAslTslCsdTsdTslGsdGslCsdCslTslC (SEQ ID NO: 695) (SEQ ID NO: 696) 4318 CTGCAGCTCCTCCA lCsdTslGsdCslAslGsdCsdTslCsdCslTsdCslCslA (SEQ ID NO: 697) (SEQ ID NO: 698) 4416 TGACTGATCAGACA lTsdGslAsdCslTslGsdAsdTslCsdAslGsdAslCslA (SEQ ID NO: 699) (SEQ ID NO: 700) 4656 ATGGCATCTGCTTC lAsdTslGsdGslCslAsdTsdCslTsdGslCsdTslTslC (SEQ ID NO: 701) (SEQ ID NO: 702) 4717 TGCCTCCTGCAGCC lTsdGslCsdCslTslCsdCsdTslGsdCslAsdGslCslC (SEQ ID NO: 703) (SEQ ID NO: 704) 4773 AGCTTGGCCTTCTC lAsdGslCsdTslTslGsdGsdCslCsdTslTsdCslTslC (SEQ ID NO: 705) (SEQ ID NO: 706) 4802 TTACATCCTCTGAC lTsdTslAsdCslAslTsdCsdCslTsdCslTsdGslAslC (SEQ ID NO: 707) (SEQ ID NO: 708) 4837 AGCAGCTGCTGAGG lAsdGslCsdAslGslCsdTsdGslCsdTslGsdAslGslG (SEQ ID NO: 709) (SEQ ID NO: 710) 4904 TCTCCTCCTCCTGC lTsdCslTsdCslCslTsdCsdCslTsdCslCsdTslGslC (SEQ ID NO: 711) (SEQ ID NO: 712) 6033 CCCTTAAGGGCCTC lCsdCslCsdTslTslAsdAsdGslGsdGslCsdCslTslC (SEQ ID NO: 713) (SEQ ID NO: 714) 6099 AGGTTCTTCCTGTC lAsdGslGsdTslTslCsdTsdTslCsdCslTsdGslTslC (SEQ ID NO: 715) (SEQ ID NO: 716) 6142 GACCTTGCTCTGCA lGsdAslCsdCslTslTsdGsdCslTsdCslTsdGslCslA (SEQ ID NO: 717) (SEQ ID NO: 718) 6149 AGCTCTTGACCTTG lAsdGslCsdTslCslTsdTsdGslAsdCslCsdTslTslG (SEQ ID NO: 719) (SEQ ID NO: 720) 6220 GTGCTGGGCCTTGC lGsdTslGsdCslTslGsdGsdGslCsdCslTsdTslGslC (SEQ ID NO: 721) (SEQ ID NO: 722) 6273 TTGTTGGCCTGGGT lTsdTslGsdTslTslGsdGsdCslCsdTslGsdGslGslT (SEQ ID NO: 723) (SEQ ID NO: 724) 6277 CAGCTTGTTGGCCT lCsdAslGsdCslTslTsdGsdTslTsdGslGsdCslCslT (SEQ ID NO: 725) (SEQ ID NO: 726)

TABLE 4 Starting  nuc1eotide of target sequence in SEQ ID NO: 6 Sequence (5′-3′) Sequence (5′-3′) with modifications 4302 +TT+GA+T+CTT+GG+ lTsdTslGsdAsLTslCsdTsdTslGsdGslCsdCslTslC CC+T+C (SEQ ID NO: 728) (SEQ ID NO: 727) 4302 +TT+GA+TC+T+T+GG lTsdTsLGsdAslTsdCslTslTslGsdGsdCslCsdTslC C+CT+C (SEQ ID NO: 730) (SEQ ID NO: 729) 4302 lTslTslGsdAsdTsmdCsdTsdTsdGsdGsmdCslCslTslC (SEQ ID NO: 731) 4302 TTGATCTTGGCCTC eTseTseGsdAsdTsmdCsdTsdTsdGsdGsmdCseCseTseC (SEQ ID NO: 732) (SEQ ID NO: 733) 4302 TTGATCTTGGCCTC lTslTsmGsdAsdTsmdCsdTsdTsdGsdGsmdCsmCslTslC (SEQ ID NO: 734) (SEQ ID NO: 735) 4302 TTGATCTTGGCCTC eTseTslGsdAsdTsmdCsdTsdTsdGsdGsmdCseCseTseC (SEQ ID NO: 736) (SEQ ID NO: 737) 4302 TTGATCTTGGCCTC eTslTslGsdAsdTsmdCsdTsdTsdGsdGsmdCslCslTslC (SEQ ID NO: 738) (SEQ ID NO: 739) 4302 TTGATCTTGGCCTC lTseTslGsdAsdTsmdCsdTsdTsdGsdGsmdCslCslTslC (SEQ ID NO: 740) (SEQ ID NO: 741) 4302 TTGATCTTGGCCTC lTslTseGsdAsdTsmdCsdTsdTsdGsdGsmdCslCslTslC (SEQ ID NO: 742) (SEQ ID NO: 743) 4302 TTGATCTTGGCCTC lTslTslGsdAsdTsmdCsdTsdTsdGsdGsmdCseCslTslC (SEQ ID NO: 744) (SEQ ID NO: 745) 4302 TTGATCTTGGCCTC lTslTslGsdAsdTsmdCsdTsdTsdGsdGsmdCslCseTslC (SEQ ID NO: 746) (SEQ ID NO: 747) 4302 TTGATCTTGGCCTC lTslTslGsdAsdTsmdCsdTsdTsdGsdGsmdCslCslTseC (SEQ ID NO: 748) (SEQ ID NO: 749) 4302 TTGATCTTGGCCTC lTslTslGdAdTmdCdTdTdGdGmdClCslTslC (SEQ ID NO: 750) (SEQ ID NO: 751) 4302 TTGATCTTGGCCTC lTslTslGsdAdTmdCdTdTdGdGmdCslCslTslC (SEQ ID NO: 752) (SEQ ID NO: 753) 4302 TTGATCTTGGCCTC lTslTslGdAsdTmdCsdTdTsdGdGsmdClCslTslC (SEQ ID NO: 754) (SEQ ID NO: 755) 4302 TTGATCTTGGCCTC lTslTlGsdAdTsmdCdTsdTdGsdGmdCslClTslC (SEQ ID NO: 756) (SEQ ID NO: 757) 4302 TTGATCTTGGCCTC lTslTslGdAsdTsmdCsdTdTsdGsdGsmdClCslTsLC (SEQ ID NO: 758) (SEQ ID NO: 759) 4318 CTGCAGCTCCTCCA lCslTsmGsmdCsdAsdGsmdCsdTsmdCsmdCsdTsmCslCslA (SEQ ID NO: 760) (SEQ ID NO: 761) 4318 CTGCAGCTCCTCCA lCslTslGmdCsdAdGsmdCdTsmdCmdCsdTlCslCslA (SEQ ID NO: 762) (SEQ ID NO: 763) 4318 CTGCAGCTCCTCCA lCslTslGmdCsdAsdGsmdCdTsmdCsmdCsdTlCslCslA (SEQ ID NO: 764) (SEQ ID NO: 765) 4302 TTGATCTTGGCCTC lTsmUslGsdAsdTsmdCsdTsdTsdGsdGsmdCslCsmuslC (SEQ ID NO: 766) (SEQ ID NO: 767) 4302 TTGATCTTGGCCTC lTslTsfGsdAsdTsmdCsdTsdTsdGsdGsmdCsfCslTslC (SEQ ID NO: 768) (SEQ ID NO: 769) 4661 lTslCsmUsdGsdGsdAsdTsdGsdGsmdCsdAsmUslCsLT (SEQ ID NO: 770)  892 lGslAsmCsdTsdGsdGsdGsdAsdGsmdCsmdCsmAslTslT (SEQ ID NO: 771) 2360 lCslCsmAsdGsdGsdAsdGsmdCsmdCsdTsdAsmUslTslC (SEQ ID NO: 772) 1417 lAslGsmAsmdCsdAsdGsdAsdAsdGsmdCsdAsmGslCslA (SEQ ID NO: 773)  516 lAslGsmUsdTsmdCsdAsmdCsdTsmdCsdAsmdCsmAslTslC (SEQ ID NO: 774) 1582 lAslAsmAsdGsdTsdGsmdCsdAsdGsdGsdAsmGslGslG (SEQ ID NO: 775) 1217 lTslCsmCsdTsmdCsdAsdGsdGsdGsdTsmdCsmUslTslG (SEQ ID NO: 776)  515 lGslTsmUsmdCsdAsmdCsdTsmdCsdAsmdCsdAsmUslCslC (SEQ ID NO: 777)  514 lTslTsmCsdAsmdCsdTsmdCsdAsmdCsdAsdTsmCslCslA (SEQ ID NO: 778) 1424 lGslGsmUsdTsmdCsdAsdTsdAsdGsdAsmdCsmAslGslA (SEQ ID NO: 779) 1219 lAslTsmUsmdCsmdCsdTsmdCsdAsdGsdGsdGsmUslCslT (SEQ ID NO: 780) 513 lTslCsmAsmdCsdTsmdCsdAsmdCsdAsdTsmdCsmCslAslT (SEQ ID NO: 781) 1226 lAslGsmUsdTsdAsdTsmdCsdAsdTsdTsmdCsmCslTslC (SEQ ID NO: 782) 1225 lGslTsmUsdAsdTsmdCsdAsdTsdTsmdCsmdCsmUslCslA (SEQ ID NO: 783) 1645 lAslCsmUsmdCsdTsmdCsdAsdGsdTsdGsmdCsmCslAslT (SEQ ID NO: 784) 4302 avTsavTsavGsdAsdTsmdCsdTsdTsdGsdGsmdCsavCsavT savC (SEQ ID NO: 785) 3257 avGsavCsavAsdCsdCsdTsdTsdGsdGsdAsdCsavTsavTsa vG (SEQ ID NO: 786) 2483 avTsavGsavAsdGsdGsdTsdTsdCsdTsdCsdCsavTsavTsa vG (SEQ ID NO: 787) l = locked nucleotide; d = deoxynucleotide; mdC = 5-Methyl cytosine; f = 2′-fluoro nucleotide; av = amino-CBBN nucleotide; s = phosphorothioate linkage; e = ethylene-bridged nucleotide

TABLE 5 Sequence (5′-3′) with modifications lGslTsdGsdAsdAsdTsdGsdCsdGsdGsdAsdTslGslAslA (SEQ ID NO: 789) lGslAslAsdGsdTsdGsdGsdTsdAsdGsdTsdCsdAslTslA (SEQ ID NO: 790) lGsdTslGsdAsdAslTslGsdCsdGslGsdAslTsdGslAslA (SEQ ID NO: 791) lGsdAslAsdGsdTslGslGsdTsdAslGsdTslCsdAslTslA (SEQ ID NO: 792)

In one embodiment, the present invention provides methods for treating or preventing a cardiac disorder in which the expression of β-MHC is up-regulated in cardiac cells of a subject comprising administering a composition comprising an inhibitor of MYH7B to the subject. In another embodiment, the present invention provides methods for treating or preventing a cardiac disorder in which the ratio of β-MHC/α-MHC is altered in cardiac cells of a subject comprising administering a composition comprising an inhibitor of MYH7B to the subject. For instance, the present invention provides compositions and methods for treating or preventing a cardiac disorder selected from the group consisting of pathologic cardiac hypertrophy, myocardial infarction, heart failure, or hypertrophic cardiomyopathy by inhibiting the expression or activity of β-MHC and/or altering the ratio of β-MHC/α-MHC in cardiac cells of a subject using an inhibitor of MYH7B. The administration of an inhibitor of MYH7B to the subject down-regulates the expression or activity of MYH7B in cardiac cells of the subject following administration. In one embodiment, the administration of an inhibitor of MYH7B does not significantly alter the expression or activity of myomiRs, particularly, miR-499, in cardiac cells of the subject. Thus, in some embodiments, the expression of miR-499, miR-208a, and/or miR-208b in cardiac cells is not statistically different after the administration of the MYH7B inhibitor.

In various embodiments, methods provided by the invention comprise administering one or more nucleic acid inhibitors disclosed herein. For instance, in one embodiment, methods provided by the invention comprise administering an antisense oligonucleotide inhibitor selected from Tables 1-5. In another embodiment, methods according to the invention comprise administering an antisense oligonucleotide inhibitor comprising the sequence of SEQ ID NO: 146. In yet another embodiment, methods according to the invention comprise administering an antisense oligonucleotide inhibitor comprising the sequence of SEQ ID NO: 148.

In a particular embodiment, the present invention provides methods for treating or preventing hypertrophic cardiomyopathy in a subject in need thereof comprising administering a composition comprising an inhibitor of MYH7B to the subject, wherein the expression or activity of MYH7B is reduced in cardiac cells of the subject following administration of the inhibitor. In one embodiment, the administration of an inhibitor of MYH7B to the subject reduces the expression or activity of β-MHC in cardiac cells of the subject following administration.

In one embodiment, the present invention provides methods for treating or preventing pathologic cardiac hypertrophy or hypertrophic cardiomyopathy in a subject at risk of developing pathologic cardiac hypertrophy or hypertrophic cardiomyopathy. The patient at risk of developing pathologic cardiac hypertrophy or hypertrophic cardiomyopathy may exhibit one or more risk factors including, for example, long standing uncontrolled hypertension, uncorrected valvular disease, chronic angina, recent myocardial infarction, congenital predisposition to heart disease or pathological hypertrophy. In some embodiments, the subject at risk may be diagnosed as having a genetic predisposition to pathologic cardiac hypertrophy or hypertrophic cardiomyopathy. For instance, in certain embodiments, the subject at risk may carry one or more mutations in sarcomeric genes, e.g., mutations in the β-MHC (MYH7) gene. In one embodiment, the subject at risk has one or more mutations in the β-MHC (MYH7) gene selected from Arg663His, Lys207Asn, Gly256Glu, Arg403Gln, Arg453Cys, Gly584Arg, Arg719Trp, Arg723Gly, Ile736Thr, Gly741Arg, Asp778Gly, Asp778Val, Asp906Gly, and Leu908Val. In certain embodiments, the subject at risk has an Arg403Gln, Arg453Cys, or Arg719Trp mutation in the β-MHC (MYH7) gene. Other mutations in sarcomeric genes associated with hypertrophic cardiomyopathy are known to those of skill in the art (see, e.g., Moore et al., Circulation Research, Vol. 111: 375-385, 2012, which hereby incorporated by reference in its entirety), and can be used to identify a subject at risk of developing pathologic cardiac hypertrophy or hypertrophic cardiomyopathy. In other particular embodiments, the subject at risk may have a familial history of pathologic cardiac hypertrophy or hypertrophic cardiomyopathy.

In another embodiment, the present invention provides compositions and methods for inhibiting the expression or activity of β-MHC in cardiac cells of a subject comprising administering an inhibitor of MYH7B to the subject. In yet another embodiment, the present invention provides compositions and methods for modulating the ratio of β-MHC/α-MHC in cardiac cells of a subject comprising administering an inhibitor of MYH7B to the subject. The administration of an inhibitor of MYH7B to the subject reduces the expression or activity of MYH7B and/or the expression or activity of β-MHC in cardiac cells of the subject compared to the expression or activity of MYH7B and β-MHC prior to the treatment with an inhibitor of MYH7B. In one embodiment, the administration of an inhibitor of MYH7B to the subject fully or partially restores the β-MHC/α-MHC ratio in cardiac cells of the subject to normal levels found in a healthy subject.

According to one aspect of the invention, administration of an inhibitor of MYH7B to the subject results in the improvement of one or more symptoms of the cardiac disorder. For instance, in one embodiment, administration of a MYH7B inhibitor results in the improvement of one or more symptoms of pathologic cardiac hypertrophy, myocardial infarction, or heart failure in the subject, or in the delay in the transition from pathologic cardiac hypertrophy to heart failure. In another embodiment, administration of a MYH7B inhibitor results in the delay in the onset of pathologic cardiac hypertrophy, myocardial infarction, or heart failure in the subject. The one or more improved symptoms may be, for example, increased exercise capacity, increased cardiac ejection volume, decreased left ventricular end diastolic pressure, decreased pulmonary capillary wedge pressure, increased cardiac output, increased cardiac index, lowered pulmonary artery pressures, decreased left ventricular end systolic and diastolic dimensions, decreased cardiac fibrosis, decreased collagen deposition in cardiac muscle, decreased left and right ventricular wall stress, decreased wall tension, increased quality of life, and decreased disease-related morbidity or mortality.

In some embodiments, an inhibitor of MYH7B is an aptamer. The term “aptamer” as used herein refers to a nucleic acid molecule having a specific binding affinity for a particular target molecule through interactions other than classic Watson-Crick base pairing. The aptamer can inhibit the activity or function of a target molecule by binding to the target molecule. In some embodiments, the aptamer of the present invention binds to one or more epitopes of the MYH7B protein. In certain embodiments, the aptamer binds to the human MYH7B protein. The anti-MYH7B aptamers may be comprised of ribonucleotides, deoxyribonucleotides, modified nucleotides, or mixtures thereof. For example, the aptamers may contain one or more 2′-O-alkyl (e.g. 2′-O-methyl) or 2′-halo (e.g. 2′-fluoro) modifications. In some embodiments the aptamer may be conjugated to a polymer, e.g. PEG polymer, to enhance the circulating half-life of the aptamer.

An aptamer for MYH7B can be identified using systematic evolution of ligands by exponential enrichment (SELEX) (Tuerk & Gold, Science 249:505-510, 1990; Ellington & Szostak, Nature 346:818-822, 1990), such as described in U.S. Pat. No. 5,270,163. A library of nucleic acids can be contacted with a target MYH7B, and those nucleic acids specifically bound to the target are partitioned from the remainder of nucleic acids in the library which do not specifically bind the target. The partitioned nucleic acids are amplified to yield a ligand-enriched pool. Multiple cycles of binding, partitioning, and amplifying (i.e., selection) result in identification of one or more aptamers with the desired activity. Modified methods, such as Laser SELEX or deSELEX as described in U.S. Patent Publication No. 20090264508 can also be used.

In certain embodiments, the length of an aptamer useful for inhibiting the expression or activity of MYH7B is about 10 to about 30 nucleotides, about 15 to about 35 nucleotides, about 20 to about 35 nucleotides, about 20 to about 40 nucleotides, or about 25 to about 50 nucleotides.

In some embodiments, an inhibitor of MYH7B is a ribozyme. The term “ribozyme” refers to an RNA molecule having catalytic activity. Ribozymes with a variety of activities are known. Alternatively, ribozymes having a target specific RNA-cleaving activity can be designed. In certain embodiments, ribozymes that can be used as an inhibitor of MYH7B include known or artificially-created RNase P ribozymes such as group I and group II intron-type ribozymes, hammerhead ribozymes and hairpin ribozymes. In some embodiments, a ribozyme that can be used as an inhibitor of MYH7B has a length of about 200 to about 500 nucleotides or about 300 to about 450 nucleotides. In other embodiments, a ribozyme inhibitor of MYH7B may have a length of about 30 to about 100 nucleotides, about 40 to about 80 nucleotides, or about 40 to about 60 nucleotides.

In one embodiment, an inhibitor of MYH7B is a small interfering RNA. As used herein, the terms “small interfering” or “short interfering RNA” or “siRNA” refer to an RNA duplex of nucleotides that is targeted to MYH7B gene and is capable of inhibiting the expression of MYH7B gene. In certain embodiments, the siRNA that may be used as an inhibitor of MYH7B comprises two complementary single-stranded RNAs of about 10 to about 30 nucleotides, particularly, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 30 nucleotides that form 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 30 base pairs and possess 5′ and/or 3′ overhangs of one or two nucleotides. The siRNA duplex comprises a first stand that has a sequence that is substantially identical to a sequence of MYH7B gene and a second strand that has a sequence that is partially, substantially, or fully complementary to the first strand. In certain embodiments, the first strand of a MYH7B siRNA comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to a sequence of MYH7B gene and the second strand comprises a sequence that is substantially or completely complementary to the first strand. In some embodiments, the first RNA strand of an siRNA targeted to MYH7B gene is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a coding region of the human MYH7B gene (SEQ ID NO: 6). In certain embodiments, the first RNA strand of an siRNA targeted to MYH7B gene is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a sequence of 5′-GAGGCCAAGATCAA-3′ (SEQ ID NO: 4). In other embodiments, the first RNA strand of an siRNA targeted to MYH7B gene is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a sequence of 5′-TGGAGGAGCTGCAG-3′ (SEQ ID NO: 5). An siRNA inhibitor of MYH7B can be administered to cardiac cells or expressed in cardiac cells from one or more vectors.

In another embodiment, an inhibitor of MYH7B is a small hairpin RNA (shRNA). As used herein, the term “shRNA” refers to an RNA duplex wherein a portion of the duplex is part of a hairpin structure (shRNA). In addition to the duplex portion, the hairpin structure may contain a loop portion positioned between the two first and the second strand that form the duplex. The loop can vary in length. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length. The hairpin structure can also contain 3′ or 5′ overhang portions. In some aspects, the overhang is a 3′ or a 5′ overhang 0, 1, 2, 3, 4 or 5 nucleotides in length. The duplex portion or double-stranded region of an shRNA targeting MYH7B is from about 10 to about 30 nucleotides in length and comprises a first RNA strand that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to a sequence of the MYH7B gene and a second RNA strand that is substantially or fully complementary to the first strand. In certain embodiments, the first RNA strand of the duplex portion of an shRNA targeted to MYH7B gene is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a coding region of the human MYH7B gene (SEQ ID NO: 6). In some embodiments, the first RNA strand of the duplex portion of an shRNA targeted to MYH7B gene is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a sequence of 5′-GAGGCCAAGATCAA-3′ (SEQ ID NO: 4). In other embodiments, the first RNA strand of an shRNA targeted to MYH7B gene is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a sequence of 5′-TGGAGGAGCTGCAG-3′ (SEQ ID NO: 5). An shRNA inhibitor of MYH7B can be administered to cardiac cells or expressed in cardiac cells from a vector.

Similar to antisense oligonucleotides, other inhibitory nucleotide molecules described herein (aptamers, ribozymes, siRNA, shRNA) may contain one or more chemically-modified nucleotides described above. For example, an inhibitory nucleotide molecule targeted to MYH7B can contain bicyclic sugar nucleoside (BSN) modifications, locked nucleic acid (LNA) modifications, sugar modifications, such as 2′-O-alkyl (e.g. 2′-O-methyl, 2′-O-methoxyethyl), 2′-halo (e.g., 2′-fluoro, 2′-chloro, 2′-bromo), and 4′ thio modifications, backbone modifications, such as one or more phosphorothioate, morpholino, or phosphonocarboxylate linkages, base modifications such as 5-methyl cytidine or 2′-4′-bridged bicyclic nucleoside modifications.

In yet another embodiment, an inhibitor of MYH7B is an antibody or a binding fragment thereof that specifically binds to one or more epitopes of MYH7B protein. For instance, in certain embodiments, an inhibitor of MYH7B is a monoclonal or polyclonal antibody that specifically binds to one or more epitopes of MYH7B protein. The monoclonal antibody may be chimeric or humanized. In another embodiment, an inhibitor of MYH7B is a fragment derived from an MYH7B-specific antibody, wherein the fragment specifically binds to one or more epitopes of MYH7B protein. Such binding fragments may include a Fab fragment, scFv fragment, or engineered antibody fragments such as a diabody, triabody, minibody or a single-domain antibody. Anti-MYH7B antibodies encompass all immunoglobulin classes, such as IgM, IgG, IgD, IgE, and IgA or their subclasses, including IgG subclasses (e.g., IgG₁, IgG₂, IgG_(2a), IgG_(2b), IgG₃, or IgG_(M)). Preferably, the anti-MYH7B antibodies or binding fragments thereof have a binding affinity for MYH7B in a range from about 1×10⁻⁷ M to about 1×10⁻¹² M, from about 1×10⁻⁸ M to about 1×10⁻¹¹ M, or from about 1×10⁻⁹ M to about 5×10⁻¹⁰ M.

As used herein, the term “subject” or “patient” refers to any vertebrate including, without limitation, humans and other primates (e.g., chimpanzees and other apes and monkey species), farm animals (e.g., cattle, sheep, pigs, goats and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like). In some embodiments, the subject is a mammal. In other embodiments, the subject is a human and an inhibitory molecule is targeted to human MYH7B gene, mRNA, pre-mRNA, or protein.

Any of the nucleic acid inhibitors of MYH7B described herein can be delivered to the target cell (e.g. cardiac cells and skeletal muscle cells) by delivering to the cell an expression vector encoding the MYH7B inhibitors. A “vector” is a composition of matter which can be used to deliver a nucleic acid of interest to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like. In one particular embodiment, the viral vector is a lentiviral vector or an adenoviral vector. An expression construct can be replicated in a living cell, or it can be made synthetically. For purposes of this application, the terms “expression construct,” “expression vector,” and “vector,” are used interchangeably to demonstrate the application of the invention in a general, illustrative sense, and are not intended to limit the invention.

In one embodiment, an expression vector for expressing an inhibitor of MYH7B comprises a promoter operably linked to a polynucleotide encoding an antisense oligonucleotide comprising a sequence of 5′-TTGATCTTGGCCTC-3′ (SEQ ID NO: 145) . In another embodiment, an expression vector for expressing an inhibitor of MYH7B comprises a promoter operably linked to a polynucleotide encoding an antisense oligonucleotide comprising a sequence of 5′-CTGCAGCTCCTCCA-3′ (SEQ ID NO: 147). The phrase “operably linked” or “under transcriptional control” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

As used herein, a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. Suitable promoters include, but are not limited to RNA pol I, pol II, pol III, and viral promoters (e.g. human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat). In one embodiment, the promoter is a tissue-specific promoter. Of particular interest are muscle specific promoters, and more particularly, endothelial and cardiac specific promoters. These include the myosin light chain-2 promoter (Franz et al. (1994) Cardioscience, Vol. 5(4):235-43; Kelly et al. (1995) J. Cell Biol., Vol. 129(2):383-396), the alpha actin promoter (Moss et al. (1996) Biol. Chem., Vol. 271(49):31688-31694), the troponin 1 promoter (Bhaysar et al. (1996) Genomics, Vol. 35(1):11-23); the Na+/Ca2+ exchanger promoter (Barnes et al. (1997) J. Biol. Chem., Vol. 272(17):11510-11517), the dystrophin promoter (Kimura et al. (1997) Dev. Growth Differ., Vol. 39(3):257-265), the alpha7 integrin promoter (Ziober and Kramer (1996) J. Bio. Chem., Vol. 271(37):22915-22), the brain natriuretic peptide promoter (LaPointe et al. (1996) Hypertension, Vol. 27(3 Pt 2):715-22) and the alpha B-crystallin/small heat shock protein promoter (Gopal-Srivastava (1995) J. Mol. Cell. Biol., Vol. 15(12):7081-7090), alpha myosin heavy chain promoter (Yamauchi-Takihara et al. (1989) Proc. Natl. Acad. Sci. USA, Vol. 86(10):3504-3508) and the ANF promoter (LaPointe et al. (1988) J. Biol. Chem., Vol. 263(19):9075-9078).

In certain embodiments, the promoter operably linked to a polynucleotide encoding a MYH7B inhibitor can be an inducible promoter. Inducible promoters are known in the art and include, but are not limited to, tetracycline promoter, metallothionein HA promoter, heat shock promoter, steroid/thyroid hormone/retinoic acid response elements, the adenovirus late promoter, and the inducible mouse mammary tumor virus LTR.

Methods of delivering expression constructs and nucleic acids to cells are known in the art and can include, for example, calcium phosphate co-precipitation, electroporation, microinjection, DEAE-dextran, lipofection, transfection employing polyamine transfection reagents, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection.

The present invention also includes pharmaceutical compositions comprising an inhibitor of MYH7B (e.g. antisense oligonucleotide, aptamer, ribozyme, siRNA, shRNA, or antibody) and a pharmaceutically acceptable carrier or excipient. For instance, the present invention provides compositions comprising an antisense oligonucleotide of MYH7B and a pharmaceutically acceptable carrier or excipient.

In one embodiment, the pharmaceutical composition comprises an effective dose of a MYH7B inhibitor and a pharmaceutically acceptable carrier. For instance, the pharmaceutical composition comprises an effective dose of an antisense oligonucleotide targeting MYH7B or an effective dose of a modified antisense oligonucleotide targeting MYH7B as described herein. In one embodiment, the pharmaceutical composition comprises an antisense oligonucleotide having a sequence of 5′-lTslTslGsdAsdTsdCsdTsdTsdGsdGsdCslCslTslC-3′ (SEQ ID NO: 146). In other embodiments, the pharmaceutical composition comprises an antisense oligonucleotide having a sequence of 5′-lCslTslGsdCsdAsdGsdCsdTsdCsdCsdTslCslCslA-3′ (SEQ ID NO: 148). In some other embodiments, the pharmaceutical composition comprises an antisense oligonucleotide a modified antisense oligonucleotide having a sequence selected from the sequences listed in Tables 1-5.

An “effective dose” is an amount sufficient to effect a beneficial or desired clinical result. An effective dose of an MYH7B inhibitor of the invention may be from about 1 mg/kg to about 100 mg/kg, about 2.5 mg/kg to about 50 mg/kg, or about 5 mg/kg to about 25 mg/kg. The precise determination of what would be considered an effective dose may be based on factors individual to each patient, including their size, age, type of disorder (e.g. hypertrophic cardiomyopathy, myocardial infarction, heart failure, or cardiac hypertrophy), and nature of inhibitor (e.g. antisense oligonucleotide, siRNA, shRNA, expression construct, antibody, etc). Therefore, dosages can be readily ascertained by those of ordinary skill in the art from this disclosure and the knowledge in the art.

In certain embodiments, the invention provides methods for treating or preventing pathologic cardiac hypertrophy, myocardial infarction, heart failure, or hypertrophic cardiomyopathy comprising administering an inhibitor of MYH7B and a second therapeutic agent. In some embodiments, the second therapeutic agent is a cardiac therapeutic agent such as an antisense oligonucleotide inhibitor of miR-208a, miR-208b, miR-499, miR-15a, miR-15b, miR-16, miR-195, or combinations thereof. Exemplary inhibitors of such miRNAs are described in WO 2012/083005 and WO 2013/192486, which are hereby incorporated by reference in their entireties. In embodiments where a second therapeutic agent is included, the second agent may be administered concurrently but in separate formulations or sequentially. In other embodiments, the second therapeutic agent may be administered at different times prior to or after administration of a MYH7B inhibitor. Prior administration includes, for instance, administration of the first agent within the range of about one week to up to 30 minutes prior to administration of the second agent. Prior administration may also include, for instance, administration of the first agent within the range of about 2 weeks to up to 30 minutes prior to administration of the second agent. After or later administration includes, for instance, administration of the second agent within the range of about one week to up to 30 minutes after administration of the first agent. After or later administration may also include, for instance, administration of the second agent within the range of about 2 weeks to up to 30 minutes after administration of the first agent.

The present invention includes pharmaceutical compositions comprising an inhibitor of MYH7B, a second therapeutic agent and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the pharmaceutical composition may comprise two or more inhibitors of MYH7B. In some embodiments, the second therapeutic agent is a cardiac therapeutic agent such as an antisense oligonucleotide inhibitor of miR-208a, miR-208b, miR-499, miR-15a, miR-15b, miR-16, miR-195, or combinations thereof. In these embodiments, the second therapeutic agent may be included in the same formulation as an inhibitor of MYH7B or in a separate formulation. Where clinical applications are contemplated, pharmaceutical compositions will be prepared in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.

Colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes, may be used as delivery vehicles for the oligonucleotide inhibitors of MYH7B, or constructs expressing MYH7B nucleotide inhibitors. Commercially available fat emulsions that are suitable for delivering the nucleic acids of the invention to cardiac and skeletal muscle tissues include Intralipid®, Liposyn®, Liposyn® II, Liposyn® III, Nutrilipid, and other similar lipid emulsions. A preferred colloidal system for use as a delivery vehicle in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art. Exemplary formulations are also disclosed in U.S. Pat. Nos. 5,981,505; 6,217,900; 6,383,512; 5,783,565; 7,202,227; 6,379,965; 6,127,170; 5,837,533; 6,747,014; and WO03/093449, which are herein incorporated by reference in their entireties.

In certain embodiments, liposomes used for delivery are amphoteric liposomes such SMARTICLES® (Marina Biotech, Inc.) which are described in detail in U.S. Pre-grant Publication No. 20110076322. The surface charge on the SMARTICLES® is fully reversible which make them particularly suitable for the delivery of nucleic acids. SMARTICLES® can be delivered via injection, remain stable, and aggregate free and cross cell membranes to deliver the nucleic acids

In some embodiments, appropriate salts and buffers are used to render delivery vehicles stable and allow for uptake by target cells. Aqueous compositions of the present invention comprise an effective amount of the delivery vehicle comprising the inhibitor polynucleotides (e.g. liposomes or other complexes or expression vectors) dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, “pharmaceutically acceptable carrier” includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate the vectors or polynucleotides of the compositions.

The active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention may be via any common route so long as the target tissue is available via that route. This includes oral, nasal (inhalational), or buccal. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or by direct injection into cardiac tissue. Pharmaceutical compositions comprising MYH7B inhibitors or expression constructs comprising MYH7B inhibitors may also be administered by catheter systems or systems that isolate coronary circulation for delivering therapeutic agents to the heart. Various catheter systems for delivering therapeutic agents to the heart and coronary vasculature are known in the art. Some non-limiting examples of catheter-based delivery methods or coronary isolation methods suitable for use in the present invention are disclosed in U.S. Pat. Nos. 6,416,510; 6,716,196; 6,953,466, WO 2005/082440, WO 2006/089340, U.S. Patent Publication No. 2007/0203445, U.S. Patent Publication No. 2006/0148742, and U.S. Patent Publication No. 2007/0060907, which are all herein incorporated by reference in their entireties. Such compositions would normally be administered as pharmaceutically acceptable compositions as described herein.

In another embodiment of the invention, compositions comprising MYH7B inhibitors as described herein may be formulated as a coating for a medical device, such as a stent, balloon, or catheter. Particularly useful in methods of treating cardiac disorders in a subject, the inhibitors of MYH7B can be used to coat a metal stent to produce a drug-eluting stent. A drug-eluting stent is a scaffold that holds open narrowed or diseased arteries and releases a compound to prevent cellular proliferation and/or inflammation. The inhibitors of MYH7B may be applied to a metal stent imbedded in a thin polymer for release of the inhibitors over time. Methods for device-based delivery and methods of coating devices are well known in the art, as are drug-eluting stents and other implantable devices. See, e.g., U.S. Pat. Nos. 7,294,329, 7,273,493, 7,247,313, 7,236,821, 7,232,573, 7,156,869, 7,144,422, 7,105,018, 7,087,263, 7,083,642, 7,055,237, 7,041,127, 6,716,242, and 6,589,286, and WO 2004/004602, which are herein incorporated by reference in their entireties. Thus, the present invention includes a medical device, such as a balloon, catheter, or stent, coated with a MYH7B inhibitor. In some embodiments, the MYH7B inhibitor can be used in combination with other therapeutic agents (e.g. anti-restenosis compounds) to produce a formulation for incorporation into drug-eluting stents.

The active compounds may also be administered parenterally or intraperitoneally. By way of illustration, solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use or catheter delivery include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that easy injectability exists. Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the active compounds in an appropriate amount into a solvent along with any other ingredients (for example as enumerated above) as desired, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients, e.g., as enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The compositions of the present invention generally may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include, for example, acid addition salts (formed with the free amino groups of the protein) derived from inorganic acids (e.g., hydrochloric or phosphoric acids), or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like). Salts formed with the free carboxyl groups of the protein can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine and the like).

Upon formulation, solutions are preferably administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations may easily be administered in a variety of dosage forms such as injectable solutions, drug release capsules, eye drops, intravitreal injections, drug-eluting stents or other coated vascular devices, and the like. For parenteral administration in an aqueous solution, for example, the solution generally is suitably buffered and the liquid diluent first rendered isotonic for example with sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous, intravitreal and intraperitoneal administration. Preferably, sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure. By way of illustration, a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory agencies.

In certain embodiments of the invention, the pharmaceutical compositions of the invention are packaged with or stored within a device for administration. Devices for injectable formulations include, but are not limited to, injection ports, autoinjectors, injection pumps, and injection pens. Devices for aerosolized or powder formulations include, but are not limited to, inhalers, insufflators, aspirators, and the like. Thus, the present invention includes administration devices comprising a pharmaceutical composition of the invention for treating or preventing one or more of the disorders described herein.

This invention is further illustrated by the following additional examples that should not be construed as limiting. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made to the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

All patent and non-patent documents referenced throughout this disclosure are incorporated by reference herein in their entirety for all purposes.

EXAMPLES Example 1 Inhibition of MYH7B Down-Regulates MYH7

Human iPS cardiomyocytes were transfected with a control oligonucleotide or antisense oligonucleotides (Anti-7b #1 (5′-GTGAATGCGGATGAA-3′; SEQ ID NO: 1) and Anti-7b #2 (5′-GAAGTGGTAGTCATA-3′; SEQ ID NO: 2) targeted to MYH7B. The levels of MYH7B and MYH7 (β-MHC) mRNA were measured at 48 hours using real-time PCR. Human iPS cardiomyocytes treated with a thyroid hormone, T3, were used as a positive control. It is known that T3 down-regulates the expression of β-MHC. The real-time PCR analysis of RNA isolated from the transfected cells showed that the inhibition of MYH7B resulted in the down-regulation of MYH7B as well as MYH7 (β-MHC) (FIG. 1). As expected, T3 down-regulated MYH7 (β-MHC) (FIG. 1B) but did not affect the levels MYH7B or miR-208a, miR-208b, and miR-499 (FIGS. 1A and 2).

The observation that MYH7B knockdown results in the myosin switch could, in part, be through the regulation of the myosin's intronic miRNAs (MYH7B, miR-499; MYH7, miR-208b; MYH6, miR-208a). Therefore, we tested whether the inhibition of MYH7B in human iPS cardiomyocytes affects the expression of the myomiRs, miR-208a, miR-208b, or miR-499.

Specifically, to determine if the down-regulation of MYH7B had any effect on the expression of myomiRs (miR-208a, miR-499, and miR-208b), the levels of miR-208a, miR-499, and miR-208b were measured 48 hours after transfection. No significant effect was observed on the expression of miR-208a, miR-499, or miR-208b (FIG. 2). Over time, there could be a down-regulation of miR-208b because of the downregulation of MYH7; however, the data in FIG. 2 shows that antisense oligonucleotides directed to MYH7B do not directly or immediately affect the expression of myomiRs. Therefore, the regulation of the myosin switch by inhibition of MYH7B represents a novel, miRNA-independent mechanism in cardiac cells.

It has been shown that miR-208a acts upstream of miR-499 and is required for post-natal miR-499, MYH7B, and β-MHC expression in rodents (van Rooji et al., “Control of stress-dependent cardiac growth and gene expression by a microRNA,” Science, 2007, 316(5824), 575-579; Montgomery et al., “Therapeutic inhibition of miR-208a improves cardiac function and survival during heart failure,” Circulation, 2011, 124(14), 1537-1547). Loss of miR-499 and MYH7B are required for β-MHC down-regulation in the heart in rodents (van Rooji et al., “A family of microRNAs encoded by myosin genes governs myosin expression and muscle performance,” Dev. Cell, 2009, 17, 662-673). Although these studies show that myomiRs play an important role in the regulation of β-MHC expression in α-MHC predominant species such as rodents, studies in β-MHC predominant species such as pigs, rabbits, and humans indicate that alternative mechanisms are likely involved in regulating the expression of β-MHC in β-MHC predominant species.

Example 2 Inhibition of MYH7B in iPS Cardiac Cells Derived from a Patient with Hypertrophic Cardiomyopathy Down-Regulates MYH7

Control iPS cardiomyocytes and iPS cardiomyocytes derived from patients with hypertrophic cardiomyopathy (HCM) having a mutation R663H were transfected with a control oligonucleotide or MYH7B antisense oligonucleotides (Anti-7b #1 and Anti-7b #2; SEQ ID NOs: 1 and 2). The levels of MYH7 (β-MHC) mRNA were measured at 48 hours and 1 week (168 hours) post-transfection using real-time PCR. The real-time PCR analysis showed that the inhibition of MYH7B resulted in the down-regulation of MYH7 (β-MHC) expression in HCM cardiomyocytes (FIG. 3A). Moreover, the down-regulation in the expression of MYH7 (β-MHC) was sustained up to 1 week post-treatment (FIG. 3B).

Example 3 Inhibition of MYH7B Down-Regulates β-MHC Protein

To determine if inhibition of MYH7B results in the down-regulation of MYH7 at the protein level, control and HCM R663H human iPS cardiomyocytes were transfected with a control oligonucleotide, MYH7B ASO #1 (SEQ ID NO: 1), MYH7B ASO and TINY. TINY is a 8mer oligo complementary to the seed regions of miR-208a, miR-208b, and miR-499. The levels of MYH7 (β-MHC) protein were assessed by western blot analysis. The results showed that the treatment of control or HCM iPS cardiomyocytes using MYH7B antisense oligonucleotides significantly down-regulated the MYH7 (β-MHC) protein at 96 hours post-treatment (FIG. 4). A control oligo showed no effect. T3 was used as a positive control.

Example 4 Screening of Antisense Oligonucleotides (ASOs) Directed to MYH7B

Human iPS cardiomyocytes were transfected with a control oligonucleotide (Mock) or 1.5 nM of test ASOs targeted to MYH7B. The levels of MYH7B mRNA were measured at 48 hours using real-time PCR (FIG. 5). A number of ASOs showed greater than 50% inhibition of MYH7B levels. Specifically, the following ASO compounds were tested:

TABLE 6 CPD # SEQ ID NO: Comp-1 SEQ ID NO: 52 Comp-2 SEQ ID NO: 8 Comp-3 SEQ ID NO: 22 Comp-4 SEQ ID NO: 82 Comp-5 SEQ ID NO: 96 Comp-6 SEQ ID NO: 110 Comp-7 SEQ ID NO: 3 Comp-8 SEQ ID NO: 140 Comp-9 SEQ ID NO: 154 Comp-10 SEQ ID NO: 170 Comp-11 SEQ ID NO: 38 Comp-12 SEQ ID NO: 54 Comp-13 SEQ ID NO: 24 Comp-14 SEQ ID NO: 68 Comp-15 SEQ ID NO: 84 Comp-16 SEQ ID NO: 98 Comp-17 SEQ ID NO: 112 Comp-18 SEQ ID NO: 126 Comp-19 SEQ ID NO: 142 Comp-20 SEQ ID NO: 156 Comp-21 SEQ ID NO: 172 Comp-22 SEQ ID NO: 56 Comp-23 SEQ ID NO: 10 Comp-24 SEQ ID NO: 26 Comp-25 SEQ ID NO: 70 Comp-26 SEQ ID NO: 86 Comp-27 SEQ ID NO: 100 Comp-28 SEQ ID NO: 114 Comp-29 SEQ ID NO: 128 Comp-30 SEQ ID NO: 144 Comp-31 SEQ ID NO: 158 Comp-32 SEQ ID NO: 174 Comp-33 SEQ ID NO: 42 Comp-34 SEQ ID NO: 58 Comp-35 SEQ ID NO: 12 Comp-36 SEQ ID NO: 28 Comp-37 SEQ ID NO: 72 Comp-38 SEQ ID NO: 88 Comp-39 SEQ ID NO: 102 Comp-40 SEQ ID NO: 116 Comp-41 SEQ ID NO: 130 Comp-42 SEQ ID NO: 146 Comp-43 SEQ ID NO: 160 Comp-44 SEQ ID NO: 176 Comp-45 SEQ ID NO: 44 Comp-46 SEQ ID NO: 60 Comp-47 SEQ ID NO: 14 Comp-48 SEQ ID NO: 30 Comp-49 SEQ ID NO: 74 Comp-50 SEQ ID NO: 90 Comp-51 SEQ ID NO: 104 Comp-52 SEQ ID NO: 118 Comp-53 SEQ ID NO: 132 Comp-54 SEQ ID NO: 148 Comp-55 SEQ ID NO: 162 Comp-56 SEQ ID NO: 178 Comp-57 SEQ ID NO: 46 Comp-58 SEQ ID NO: 62 Comp-59 SEQ ID NO: 16 Comp-60 SEQ ID NO: 32 Comp-61 SEQ ID NO: 76 Comp-62 SEQ ID NO: 92 Comp-63 SEQ ID NO: 3 Comp-64 SEQ ID NO: 120 Comp-65 SEQ ID NO: 134 Comp-66 SEQ ID NO: 150 Comp-67 SEQ ID NO: 164 Comp-68 SEQ ID NO: 180 Comp-69 SEQ ID NO: 48 Comp-70 SEQ ID NO: 64 Comp-71 SEQ ID NO: 18 Comp-72 SEQ ID NO: 34 Comp-73 SEQ ID NO: 78 Comp-74 SEQ ID NO: 106 Comp-75 SEQ ID NO: 122 Comp-76 SEQ ID NO: 136 Comp-77 SEQ ID NO: 152 Comp-78 SEQ ID NO: 166 Comp-79 SEQ ID NO: 182 Comp-80 SEQ ID NO: 50 Comp-81 SEQ ID NO: 66 Comp-82 SEQ ID NO: 36 Comp-83 SEQ ID NO: 80 Comp-84 SEQ ID NO: 94 Comp-85 SEQ ID NO: 108 Comp-86 SEQ ID NO: 124 Comp-87 SEQ ID NO: 138 Comp-88 SEQ ID NO: 168

Example 5 Inhibition of MYH7B Upon Passive Administration of ASOs

ASOs that showed more than 50% inhibition of MYH7B levels in Example 4 were further tested for their inhibitory potential upon passive uptake. Specifically, selected ASOs were incubated with human iPS cardiomyocytes for 168 hours at 1, 5, or 10 μM ASO concentrations. The levels of MYH7B, MYH7 (β-MHC), and MYH6 (α-MHC) mRNAs were measured at 168 hours using real-time PCR. The real-time PCR analysis showed that many ASOs downregulated MYH7B mRNA levels (FIG. 6A). Moreover, antisense oligonucleotide having the sequence of SEQ ID NO: 146 showed a “switch” in the levels of myosins, where β-MHC is down-regulated and α-MHC is up-regulated following inhibition of MYH7B (FIGS. 6B and 6C). Some other ASOs also showed a similar trend in the levels of β- and α-MHC (FIGS. 6B and 6C).

Another ASO compound having the sequence of SEQ ID NO: 146, was also tested for its inhibitory activity upon passive uptake. Human iPS cardiomyocytes were incubated with 5 μM concentration of the compound with SEQ ID NO: 146 for 168 hours and the levels of MYH7B, MYH7 (β-MHC), and MYH6 (α-MHC) mRNAs were measured using real-time PCR. As shown in FIG. 7, the compound with SEQ ID NO: 146 also showed a “switch” in the levels of myosins, where β-MHC is down-regulated and α-MHC is up-regulated following inhibition of MYH7B.

Example 6 In Vivo Activity of Test ASOs

ASOs that showed in vitro inhibition of MYH7B were further tested for their inhibitory potential in vivo. Specifically, selected ASOs were injected subcutaneously into rats at a dose of 25 mg/kg per day for three consecutive days and rats were sacrificed 48 hours after the last dose. The levels of MYH7B mRNA in left ventricles of rats were measured using real-time PCR (FIG. 8). As shows in FIG. 8, antisense oligonucleotide compounds that showed robust activity via passive administration (e.g. SEQ ID NO: 146) also showed downregulation of MYH7B in vivo, whereas compounds that showed no activity via passive administration (e.g. SEQ ID NO: 32) also showed no activity in vivo.

Compounds having the sequence of SEQ ID NOs: 146 and 148 that showed robust activity in rats were selected for rabbit studies for assessment in β-MHC-predominant species. Rabbits were injected with 10, 15, 20, or 25 mg/kg of compounds with SEQ ID NOs: 146 and 148 at days 1, 3, 5, 10, and 17 subcutaneously and sacrificed at day 18. Real-time PCR data for MYH7B shows a dose-dependent reduction in MYH7B in both ventricles (FIGS. 9A-9B). Further, the rabbit with the most robust inhibition of MYH7B (5003) also showed the most robust reduction in β-MHC (MYH7). See FIGS. 9C and 9D.

Example 7 Effect of Nucleotide Modifications on the Activity of MYH7B ASOs

The ASO compound with SEQ ID NO: 146 was modified at various positions to assess the effect of modifications on its activity. Modifications included substitution of LNA nucleotides with corresponding ENA nucleotides and other base and sugar modifications. FIG. 10A shows that substitution of ENA for LNA retained passive activity targeting MYH7B. Likewise, substitution of cytidine with 5′-methyl cytidine and substitution of an LNA base with 2′O-methyl modified nucleotide retain activity (FIG. 10B). FIG. 10C shows backbone modifications greatly affect the passive activity of compounds.

Example 8 Screening of Additional ASOs

Additional antisense oligonucleotide compounds were screened for their inhibitory potential upon passive administration. Human iPS cardiomyocytes were passively treated with test compounds at 5 μM for 48 hours. The levels of MYH7B mRNA were measured at 48 hours using real-time PCR. A number of ASOs showed robust inhibition of MYH7B levels whereas other compounds showed no effect on MYH7B levels. FIGS. 11A-11D show results from 4 separate studies. Specifically, the following ASO compounds were tested.

TABLE 7 CPD # SEQ ID NO: Comp-1 SEQ ID NO: 184 Comp-2 SEQ ID NO: 186 Comp-3 SEQ ID NO: 188 Comp-4 SEQ ID NO: 190 Comp-5 SEQ ID NO: 192 Comp-6 SEQ ID NO: 194 Comp-7 SEQ ID NO: 196 Comp-8 SEQ ID NO: 198 Comp-9 SEQ ID NO: 200 Comp-10 SEQ ID NO: 202 Comp-11 SEQ ID NO: 204 Comp-12 SEQ ID NO: 206 Comp-13 SEQ ID NO: 208 Comp-14 SEQ ID NO: 210 Comp-15 SEQ ID NO: 212 Comp-16 SEQ ID NO: 214 Comp-17 SEQ ID NO: 216 Comp-18 SEQ ID NO: 218 Comp-19 SEQ ID NO: 220 Comp-20 SEQ ID NO: 222 Comp-21 SEQ ID NO: 224 Comp-22 SEQ ID NO: 226 Comp-23 SEQ ID NO: 228 Comp-24 SEQ ID NO: 230 Comp-25 SEQ ID NO: 232 Comp-26 SEQ ID NO: 234 Comp-27 SEQ ID NO: 236 Comp-28 SEQ ID NO: 238 Comp-29 SEQ ID NO: 240 Comp-30 SEQ ID NO: 242 Comp-31 SEQ ID NO: 244 Comp-32 SEQ ID NO: 246 Comp-33 SEQ ID NO: 248 Comp-34 SEQ ID NO: 250 Comp-35 SEQ ID NO: 252 Comp-36 SEQ ID NO: 254 Comp-37 SEQ ID NO: 256 Comp-38 SEQ ID NO: 258 Comp-39 SEQ ID NO: 260 Comp-40 SEQ ID NO: 262 Comp-41 SEQ ID NO: 264 Comp-42 SEQ ID NO: 266 Comp-43 SEQ ID NO: 268 Comp-44 SEQ ID NO: 270 Comp-45 SEQ ID NO: 272 Comp-46 SEQ ID NO: 274 Comp-47 SEQ ID NO: 276 Comp-48 SEQ ID NO: 278 Comp-49 SEQ ID NO: 280 Comp-50 SEQ ID NO: 282 Comp-51 SEQ ID NO: 284 Comp-52 SEQ ID NO: 286 Comp-53 SEQ ID NO: 288 Comp-54 SEQ ID NO: 290 Comp-55 SEQ ID NO: 292 Comp-56 SEQ ID NO: 294 Comp-57 SEQ ID NO: 296 Comp-58 SEQ ID NO: 298 Comp-59 SEQ ID NO: 300 Comp-60 SEQ ID NO: 302 Comp-61 SEQ ID NO: 304 Comp-62 SEQ ID NO: 306 Comp-63 SEQ ID NO: 308 Comp-64 SEQ ID NO: 310 Comp-65 SEQ ID NO: 312 Comp-66 SEQ ID NO: 314 Comp-67 SEQ ID NO: 330 Comp-68 SEQ ID NO: 342 Comp-69 SEQ ID NO: 320 Comp-70 SEQ ID NO: 332 Comp-71 SEQ ID NO: 344 Comp-72 SEQ ID NO: 322 Comp-73 SEQ ID NO: 334 Comp-74 SEQ ID NO: 346 Comp-75 SEQ ID NO: 324 Comp-76 SEQ ID NO: 336 Comp-77 SEQ ID NO: 348 Comp-78 SEQ ID NO: 326 Comp-79 SEQ ID NO: 338 Comp-80 SEQ ID NO: 350 Comp-81 SEQ ID NO: 328 Comp-82 SEQ ID NO: 340 Comp-83 SEQ ID NO: 362 Comp-84 SEQ ID NO: 374 Comp-85 SEQ ID NO: 352 Comp-86 SEQ ID NO: 364 Comp-87 SEQ ID NO: 376 Comp-88 SEQ ID NO: 354 Comp-89 SEQ ID NO: 366 Comp-90 SEQ ID NO: 378 Comp-91 SEQ ID NO: 356 Comp-92 SEQ ID NO: 368 Comp-93 SEQ ID NO: 380 Comp-94 SEQ ID NO: 358 Comp-95 SEQ ID NO: 370 Comp-96 SEQ ID NO: 382 Comp-97 SEQ ID NO: 360 Comp-98 SEQ ID NO: 372 Comp-99 SEQ ID NO: 394 Comp-100 SEQ ID NO: 406 Comp-101 SEQ ID NO: 384 Comp-102 SEQ ID NO: 396 Comp-103 SEQ ID NO: 408 Comp-104 SEQ ID NO: 386 Comp-105 SEQ ID NO: 398 Comp-106 SEQ ID NO: 410 Comp-107 SEQ ID NO: 388 Comp-108 SEQ ID NO: 400 Comp-109 SEQ ID NO: 412 Comp-110 SEQ ID NO: 390 Comp-111 SEQ ID NO: 402 Comp-112 SEQ ID NO: 414 Comp-113 SEQ ID NO: 392 Comp-114 SEQ ID NO: 404 Comp-115 SEQ ID NO: 426 Comp-116 SEQ ID NO: 416 Comp-117 SEQ ID NO: 316 Comp-118 SEQ ID NO: 418 Comp-119 SEQ ID NO: 318 Comp-120 SEQ ID NO: 420 Comp-121 SEQ ID NO: 422 Comp-122 SEQ ID NO: 424 Comp-123 SEQ ID NO: 428 Comp-124 SEQ ID NO: 430 Comp-125 SEQ ID NO: 432 Comp-126 SEQ ID NO: 434 Comp-127 SEQ ID NO: 436 Comp-128 SEQ ID NO: 438 Comp-129 SEQ ID NO: 440 Comp-130 SEQ ID NO: 442 Comp-131 SEQ ID NO: 444 Comp-132 SEQ ID NO: 446 Comp-133 SEQ ID NO: 448 Comp-134 SEQ ID NO: 450 Comp-135 SEQ ID NO: 452 Comp-136 SEQ ID NO: 454 Comp-137 SEQ ID NO: 456 Comp-138 SEQ ID NO: 458 Comp-139 SEQ ID NO: 460 Comp-140 SEQ ID NO: 462 Comp-141 SEQ ID NO: 464 Comp-142 SEQ ID NO: 466 Comp-143 SEQ ID NO: 468 Comp-144 SEQ ID NO: 470 Comp-145 SEQ ID NO: 472 Comp-146 SEQ ID NO: 474 Comp-147 SEQ ID NO: 476 Comp-148 SEQ ID NO: 478 Comp-149 SEQ ID NO: 480 Comp-150 SEQ ID NO: 482 Comp-151 SEQ ID NO: 484 Comp-152 SEQ ID NO: 486 Comp-153 SEQ ID NO: 488 Comp-154 SEQ ID NO: 490 Comp-155 SEQ ID NO: 492 Comp-156 SEQ ID NO: 494 Comp-157 SEQ ID NO: 496 Comp-158 SEQ ID NO: 498 Comp-159 SEQ ID NO: 500 Comp-160 SEQ ID NO: 502 Comp-161 SEQ ID NO: 504 Comp-162 SEQ ID NO: 506 Comp-163 SEQ ID NO: 508 Comp-164 SEQ ID NO: 510 Comp-165 SEQ ID NO: 512 Comp-166 SEQ ID NO: 514 Comp-167 SEQ ID NO: 516 Comp-168 SEQ ID NO: 518 Comp-169 SEQ ID NO: 522 Comp-170 SEQ ID NO: 544 Comp-171 SEQ ID NO: 524 Comp-172 SEQ ID NO: 534 Comp-173 SEQ ID NO: 546 Comp-174 SEQ ID NO: 526 Comp-175 SEQ ID NO: 536 Comp-176 SEQ ID NO: 548 Comp-177 SEQ ID NO: 528 Comp-178 SEQ ID NO: 538 Comp-179 SEQ ID NO: 550 Comp-180 SEQ ID NO: 530 Comp-181 SEQ ID NO: 540 Comp-182 SEQ ID NO: 552 Comp-183 SEQ ID NO: 532 Comp-184 SEQ ID NO: 542 Comp-185 SEQ ID NO: 554 Comp-186 SEQ ID NO: 576 Comp-187 SEQ ID NO: 556 Comp-188 SEQ ID NO: 566 Comp-189 SEQ ID NO: 578 Comp-190 SEQ ID NO: 558 Comp-191 SEQ ID NO: 568 Comp-192 SEQ ID NO: 580 Comp-193 SEQ ID NO: 560 Comp-194 SEQ ID NO: 570 Comp-195 SEQ ID NO: 582 Comp-196 SEQ ID NO: 562 Comp-197 SEQ ID NO: 572 Comp-198 SEQ ID NO: 584 Comp-199 SEQ ID NO: 564 Comp-200 SEQ ID NO: 574 Comp-201 SEQ ID NO: 586 Comp-202 SEQ ID NO: 598 Comp-203 SEQ ID NO: 588 Comp-204 SEQ ID NO: 600 Comp-205 SEQ ID NO: 610 Comp-206 SEQ ID NO: 590 Comp-207 SEQ ID NO: 602 Comp-208 SEQ ID NO: 612 Comp-209 SEQ ID NO: 592 Comp-210 SEQ ID NO: 604 Comp-211 SEQ ID NO: 614 Comp-212 SEQ ID NO: 594 Comp-213 SEQ ID NO: 606 Comp-214 SEQ ID NO: 616 Comp-215 SEQ ID NO: 596 Comp-216 SEQ ID NO: 608 Comp-217 SEQ ID NO: 618 Comp-218 SEQ ID NO: 620 Comp-219 SEQ ID NO: 622 Comp-220 SEQ ID NO: 624 Comp-221 SEQ ID NO: 626 Comp-222 SEQ ID NO: 136 Comp-223 SEQ ID NO: 146 Comp-224 SEQ ID NO: 148 Comp-225 SEQ ID NO: 520 

The invention claimed is:
 1. An antisense oligonucleotide inhibitor of MYH7B containing at least one modified nucleotide, wherein the antisense oligonucleotide is 8 to 18 nucleotides and wherein the sequence of the antisense oligonucleotide is completely complementary to a sequence of SEQ ID NO: 6 from nucleotides 4300-4317, nucleotides 4302-4315, or nucleotides 4318-4331.
 2. The antisense oligonucleotide inhibitor of claim 1, wherein the antisense oligonucleotide is 14 nucleotides and the sequence of the antisense oligonucleotide is completely complementary to a sequence of SEQ ID NO: 6 from nucleotides 4300-4317.
 3. The antisense oligonucleotide inhibitor of claim 1, wherein the antisense oligonucleotide is 14 nucleotides and the sequence of the antisense oligonucleotide is completely complementary to the sequence of SEQ ID NO: 6 from nucleotides 4302-4315.
 4. The antisense oligonucleotide inhibitor of claim 1, wherein the antisense oligonucleotide is 14 nucleotides and the sequence of the antisense oligonucleotide is completely complementary to the sequence of SEQ ID NO: 6 from nucleotides 4318-4331.
 5. The antisense oligonucleotide inhibitor of claim 1, wherein said modified nucleotide includes a sugar, base, or a backbone modification.
 6. The antisense oligonucleotide inhibitor of claim 5, wherein the sugar modification is selected from the group consisting of 2′-O, 4′-C methylene bridge, 2′-O, 4′-C ethylene bridge, 2′-CH₂—NH—CH₂-4′ bridge, 2′-deoxy, 2′-O-alkyl, and 2′-halo modifications.
 7. The antisense oligonucleotide inhibitor of claim 5, wherein the backbone modification is a phosphorothioate linkage.
 8. The antisense oligonucleotide inhibitor of claim 7, wherein said antisense oligonucleotide contains two or more phosphorothioate linkages.
 9. The antisense oligonucleotide inhibitor of claim 8, wherein said antisense oligonucleotide is fully phosphorothioate linked.
 10. The antisense oligonucleotide inhibitor of claim 1, wherein said modified nucleotide is a locked nucleotide.
 11. The antisense oligonucleotide inhibitor of claim 1, wherein said antisense oligonucleotide contains one to six locked nucleotides.
 12. The antisense oligonucleotide inhibitor of claim 1, wherein said antisense oligonucleotide contains at least three locked nucleotides at the 5′ end.
 13. The antisense oligonucleotide inhibitor of claim 12, wherein the at least three locked nucleotides at the 5′ end are ribonucleotides.
 14. The antisense oligonucleotide inhibitor of claim 1, wherein said antisense oligonucleotide contains at least three locked nucleotides at the 3′ end.
 15. The antisense oligonucleotide inhibitor of claim 14, wherein the at least three locked nucleotides at the 3′ end are ribonucleotides.
 16. The antisense oligonucleotide inhibitor of claim 1, wherein said antisense oligonucleotide contains at least one deoxyribonucleotide.
 17. The antisense oligonucleotide inhibitor of claim 16, wherein said antisense oligonucleotide contains two to eight deoxyribonucleotides.
 18. The antisense oligonucleotide inhibitor of claim 1, wherein said modified nucleotide is 5′-methyl cytidine.
 19. A pharmaceutical composition comprising the antisense oligonucleotide inhibitor of MYH7B of claim 1 and a pharmaceutically acceptable excipient.
 20. The pharmaceutical composition of claim 19, further comprising a second therapeutic agent, wherein the second therapeutic agent is an antisense oligonucleotide inhibitor of miR-208a, miR-208b, miR-499, miR-15a, miR-15b, miR-16, miR-195, or a mixture thereof.
 21. An antisense oligonucleotide inhibitor of MYH7B containing at least one modified nucleotide, and wherein said antisense oligonucleotide comprises a sequence selected from Tables 1-5.
 22. The antisense oligonucleotide inhibitor of claim 21, wherein said antisense oligonucleotide comprises the sequence of 5′-lTslTslGsdAsdTsdCsdTsdTsdGsdGsdCslCslTslC-3′ (SEQ ID NO: 146) or 5′ -lCslTslGsdCsdAsdGsdCsdTsdCsdCsdTslCslCslA-3′ (SEQ ID NO: 148).
 23. The antisense oligonucleotide inhibitor of claim 21, wherein said antisense oligonucleotide comprises the sequence of 5′-lCslCslAsdGsdGsdAsdGsmdCsmdCsdTsdAslTslTslC-3′ (SEQ ID NO: 494).
 24. The antisense oligonucleotide inhibitor of claim 21, wherein said antisense oligonucleotide comprises the sequence of 5′-lGslTslTsmdCsdAsmdCsdTsmdCsdAsmdCsdAslTslCslC-3′ (SEQ ID NO: 188).
 25. The antisense oligonucleotide inhibitor of claim 21, wherein said antisense oligonucleotide comprises the sequence of 5′-lAslGslTsdTsmdCsdAsmdCsdTsmdCsdAsmdCslAslTslC-3′ (SEQ ID NO: 190).
 26. The antisense oligonucleotide inhibitor of claim 21, wherein said antisense oligonucleotide comprises the sequence of 5′-lAslGslTsdTsdAsdTsmdCsdAsdTsdTsmdCslCslTslC-3′ (SEQ ID NO: 370).
 27. The antisense oligonucleotide inhibitor of claim 21, wherein said antisense oligonucleotide comprises the sequence of 5′-lCslTslTsdAsdGsmdCsdTsdGsdAsdTsmdCslCslTslC-3′ (SEQ ID NO: 568).
 28. A pharmaceutical composition comprising the antisense oligonucleotide inhibitor of MYH7B of claim 21 and a pharmaceutically acceptable excipient.
 29. The pharmaceutical composition of claim 28, further comprising a second therapeutic agent, wherein the second therapeutic agent is an antisense oligonucleotide inhibitor of miR-208a, miR-208b, miR-499, miR-15a, miR-15b, miR-16, miR-195, or a mixture thereof. 